Combination therapy for treating cancer

ABSTRACT

The present invention relates to compositions comprising inhibitors of human histone methyltransferase EZH2 and one or more other therapeutic agents, particularly anticancer agents such as prednisone, and methods of combination therapy for administering to subjects in need thereof for the treatment of cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/394,463 filed Oct. 14, 2014 (now allowed), which is a U.S. NationalPhase application of International Application No. PCT/US2013/036452,filed Apr. 12, 2013, which claims priority to, and the benefit of, theU.S. Provisional Application No. 61/624,194 filed Apr. 13, 2012 and theU.S. Provisional Application No. 61/785,169 filed Mar. 14, 2013, thecontents of each of which are incorporated by reference in theirentireties.

PARTIES TO JOINT RESEARCH AGREEMENT

This invention was developed subject to a Joint Research Agreementbetween Epizyme, Inc. and Eisai Co., Ltd.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “EPIZ009001WO_ST25.txt,” which wascreated on Oct. 14, 2014 and is 45 KB in size, are hereby incorporatedby reference in their entireties.

FIELD OF THE INVENTION

This invention relates to compositions comprising inhibitors of humanhistone methyltransferase EZH2, the catalytic subunit of the PRC2complex which catalyzes the mono-through tri-methylation of lysine 27 onhistone H3 (H3-K27), and one or more other therapeutic agents,particularly anticancer agents, and methods of combination therapy fortreating cancer.

BACKGROUND OF THE INVENTION

Combination-therapy treatments for cancer have become more common, inpart due to the perceived advantage of attacking the disease viamultiple avenues. Although many effective combination-therapy treatmentshave been identified over the past few decades; in view of thecontinuing high number of deaths each year resulting from cancer, acontinuing need exists to identify effective therapeutic regimens foruse in anticancer treatment.

SUMMARY OF THE INVENTION

In one aspect, the present invention features a composition comprising acompound of Formula (IIa) below and one or more other therapeutic agentsor a pharmaceutically acceptable salt or ester thereof.

The compounds of Formula (IIa) can include one or more of the followingfeatures:

Each of R_(a) and R_(b), independently is H or C₁-C₆ alkyl.

R_(a) and R_(b), together with the N atom to which they are attached, isa 4 to 7-membered heterocycloalkyl ring having 0 or 1 additionalheteroatom, the C₁-C₆ alkyl and the 4 to 12-membered (e.g., 4 to7-membered) heterocycloalkyl ring being optionally substituted with oneor more -Q₃-T₃.

Q₃ is a bond or unsubstituted or substituted C₁-C₃ alkyl linker.

T₃ is H, halo, 4 to 7-membered heterocycloalkyl, C₁-C₃ alkyl, OR_(d),COOR_(d), —S(O)₂R_(d), or —NR_(d)R_(e), each of R_(d) and R_(e)independently being H or C₁-C₆ alkyl.

R₇ is C₁-C₆ alkyl, C₃-C₅ cycloalkyl or 4 to 12-membered (e.g., 4 to7-membered) heterocycloalkyl, each optionally substituted with one ormore -Q₅-T₅. For example, R₇ is not H.

R₇ is 4 to 7-membered heterocycloalkyl optionally substituted with oneor more -Q₅-T₅.

R₇ is piperidinyl, tetrahydropyran, cyclopentyl, or cyclohexyl, eachoptionally substituted with one -Q₅-T₅.

T₅ is H, halo, C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, or 4 to 12-membered (e.g., 4 to 7-membered) heterocycloalkyl.

Q₅ is a bond and T₅ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, or 4 to12-membered (e.g., 4 to 7-membered) heterocycloalkyl.

Q₅ is CO, S(O)₂, or NHC(O); and T₅ is C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₃-C₈cycloalkyl, or 4 to 12-membered (e.g., 4 to 7-membered)heterocycloalkyl.

Q₅ is C₁-C₃ alkyl linker and T₅ is H or C₆-C₁₀ aryl.

Q₅ is C₁-C₃ alkyl linker and T₅ is C₃-C₈ cycloalkyl, 4 to 7-memberedheterocycloalkyl, or S(O)_(q)R_(q).

R₇ is cyclopentyl or cyclohexyl, each optionally substituted with one-Q₅-T₅.

Q₅ is NHC(O) and T₅ is C₁-C₆ alkyl or C₁-C₆ alkoxy.

R₇ is isopropyl.

Each of R₂ and R₄, independently is H or C₁-C₆ alkyl optionallysubstituted with amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, orC₆-C₁₀ aryl.

R₈ is H, methyl, or ethyl.

R₈ is methyl.

R₈ is ethyl.

R₈ is 4 to 7-heterocycloalkyl, e.g., tetrahydropyran.

The present invention features a composition comprising a compoundselected from Table 1 or a pharmaceutically acceptable salt or esterthereof and one or more other therapeutic agents.

The present invention features a composition comprising Compound 44

or a pharmaceutically acceptable salt or ester thereof and one or moreother therapeutic agents.

In this and other aspects of the invention, in one embodiment the othertherapeutic agents are anticancer agents.

In this and other aspects of the invention, in one embodiment the othertherapeutic agents are glucocorticoids.

In this and other aspects of the invention, in one embodiment the othertherapeutic agents are selected from prednisone, prednisolone,cyclophosphamide, vincristine, doxorubicin, mafosfamide, cisplatin,AraC, everolimus, decitabine, dexamethasone, and analogs, derivatives,or combinations thereof.

In this and other aspects of the invention, in one embodiment the othertherapeutic agent is prednisone, or an analog or derivative thereof.

The present invention also provides pharmaceutical compositionscomprising a compound selected from those of Formula (IIa) disclosedherein or pharmaceutical acceptable salts thereof and one or moretherapeutic agents, and a pharmaceutically acceptable carrier.

The present invention also provides pharmaceutical compositionscomprising a compound selected from Table I, one or more othertherapeutic agents, or pharmaceutically acceptable salts thereof, and apharmaceutically acceptable carrier.

The present invention also provides pharmaceutical compositionscomprising a compound selected from those of Formula (IIa) disclosedherein or pharmaceutically acceptable salts thereof, one or more othertherapeutic agents, and a pharmaceutically acceptable carrier.

Another aspect of this invention is a method for treating or preventinga disease by administering to a subject in need thereof atherapeutically effective amount of a composition comprising a compoundof Formula (IIa), or a pharmaceutically acceptable salt thereof, and oneor more additional therapeutic agents. The disease of the presentinvention can be influenced, treated, or prevented by modulating themethylation status of histones or other proteins. For example, thedisease is cancer, a precancerous condition, or a neurological disease.Preferably, the lymphoma is non-Hodgkin lymphoma, follicular lymphoma ordiffuse large B-cell lymphoma. Alternatively, the leukemia is chronicmyelogenous leukemia (CML). The precancerous condition is, e.g.,myelodysplastic syndromes (MDS, formerly known as preleukemia).

The subject of the present invention includes any human subject who hasbeen diagnosed with, has symptoms of, or is at risk of developing acancer or a precancerous condition. The subject of the present inventionincludes any human subject expressing a mutant EZH2. For example, amutant EZH2 comprises one or more mutations, wherein the mutation is asubstitution, a point mutation, a nonsense mutation, a missensemutation, a deletion, or an insertion. A mutant EZH2 of the presentinvention may comprise a mutation in the substrate pocket domain asdefined in SEQ ID NO: 6. A mutant EZH2 may have a substitution at aminoacid Y641. Preferably, the mutant EZH2 has one of the followingmutations: substitution of phenylalanine (F) for the wild type residuetyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641F); asubstitution of histidine (H) for the wild type residue tyrosine (Y) atamino acid position 641 of SEQ ID NO: 1 (Y641H); a substitution ofasparagine (N) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641N); a substitution of serine (S) forthe wild type residue tyrosine (Y) at amino acid position 641 of SEQ IDNO: 1 (Y641S); and a substitution of cysteine (C) for the wild typeresidue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641C).

Other mutations of EZH2 may include, but are not limited to: asubstitution of glycine (G) for the wild type residue alanine (A) atamino acid position 677 of SEQ ID NO: 1 (A677G); a substitution ofvaline (V) for the wild type residue alanine (A) at amino acid position687 of SEQ ID NO: 1 (A687V); a substitution of methionine (M) for thewild type residue valine (V) at amino acid position 674 of SEQ ID NO: 1(V674M); a substitution of histidine (H) for the wild type residuearginine (R) at amino acid position 685 of SEQ ID NO: 1 (R685H); asubstitution of cysteine (C) for the wild type residue arginine (R) atamino acid position 685 of SEQ ID NO: 1 (R685C); a substitution ofserine (S) for the wild type residue asparagine (N) at amino acidposition 322 of SEQ ID NO: 3 (N322S), a substitution of glutamine (Q)for the wild type residue arginine (R) at amino acid position 288 of SEQID NO: 3 (R288Q), a substitution of isoleucine (I) for the wild typeresidue threonine (T) at amino acid position 573 of SEQ ID NO: 3(T573I), a substitution of glutamic acid (E) for the wild type residueaspartic acid (D) at amino acid position 664 of SEQ ID NO: 3 (D664E), asubstitution of glutamine (Q) for the wild type residue arginine (R) atamino acid position 458 of SEQ ID NO: 5 (R458Q), a substitution oflysine (K) for the wild type residue glutamic acid (E) at amino acidposition 249 of SEQ ID NO: 3 (E249K), a substitution of cysteine (C) forthe wild type residue arginine (R) at amino acid position 684 of SEQ IDNO: 3 (R684C), a substitution of histidine (H) for the wild type residuearginine (R) at amino acid position 628 of SEQ ID NO: 11 (R628H), asubstitution of histidine (H) for the wild type residue glutamine (Q) atamino acid position 501 of SEQ ID NO: 5 (Q501H), a substitution ofasparagine (N) for the wild type residue aspartic acid (D) at amino acidposition 192 of SEQ ID NO: 3 (D192N), a substitution of valine (V) forthe wild type residue aspartic acid (D) at amino acid position 664 ofSEQ ID NO: 3 (D664V), a substitution of leucine (L) for the wild typeresidue valine (V) at amino acid position 704 of SEQ ID NO: 3 (V704L), asubstitution of serine (S) for the wild type residue proline (P) atamino acid position 132 of SEQ ID NO: 3 (P132S), a substitution oflysine (K) for the wild type residue glutamic acid (E) at amino acidposition 669 of SEQ ID NO: 11 (E669K), a substitution of threonine (T)for the wild type residue alanine (A) at amino acid position 255 of SEQID NO: 3 (A255T), a substitution of valine (V) for the wild type residueglutamic acid (E) at amino acid position 726 of SEQ ID NO: 3 (E726V), asubstitution of tyrosine (Y) for the wild type residue cysteine (C) atamino acid position 571 of SEQ ID NO: 3 (C571Y), a substitution ofcysteine (C) for the wild type residue phenylalanine (F) at amino acidposition 145 of SEQ ID NO: 3 (F145C), a substitution of threonine (T)for the wild type residue asparagine (N) at amino acid position 693 ofSEQ ID NO: 3 (N693T), a substitution of serine (S) for the wild typeresidue phenylalanine (F) at amino acid position 145 of SEQ ID NO: 3(F145S), a substitution of histidine (H) for the wild type residueglutamine (Q) at amino acid position 109 of SEQ ID NO: 11 (Q109H), asubstitution of cysteine (C) for the wild type residue phenylalanine (F)at amino acid position 622 of SEQ ID NO: 11 (F622C), a substitution ofarginine (R) for the wild type residue glycine (G) at amino acidposition 135 of SEQ ID NO: 3 (G135R), a substitution of glutamine (Q)for the wild type residue arginine (R) at amino acid position 168 of SEQID NO: 5 (R168Q), a substitution of arginine (R) for the wild typeresidue glycine (G) at amino acid position 159 of SEQ ID NO: 3 (G159R),a substitution of cysteine (C) for the wild type residue arginine (R) atamino acid position 310 of SEQ ID NO: 5 (R310C), a substitution ofhistidine (H) for the wild type residue arginine (R) at amino acidposition 561 of SEQ ID NO: 3 (R561H), a substitution of histidine (H)for the wild type residue arginine (R) at amino acid position 634 of SEQID NO: 11 (R634H), a substitution of arginine (R) for the wild typeresidue glycine (G) at amino acid position 660 of SEQ ID NO: 3 (G660R),a substitution of cysteine (C) for the wild type residue tyrosine (Y) atamino acid position 181 of SEQ ID NO: 3 (Y181C), a substitution ofarginine (R) for the wild type residue histidine (H) at amino acidposition 297 of SEQ ID NO: 3 (H297R), a substitution of serine (S) forthe wild type residue cysteine (C) at amino acid position 612 of SEQ IDNO: 11 (C612S), a substitution of tyrosine (Y) for the wild type residuehistidine (H) at amino acid position 694 of SEQ ID NO: 3 (H694Y), asubstitution of alanine (A) for the wild type residue aspartic acid (D)at amino acid position 664 of SEQ ID NO: 3 (D664A), a substitution ofthreonine (T) for the wild type residue isoleucine (I) at amino acidposition 150 of SEQ ID NO: 3 (I150T), a substitution of arginine (R) forthe wild type residue isoleucine (I) at amino acid position 264 of SEQID NO: 3 (I264R), a substitution of leucine (L) for the wild typeresidue proline (P) at amino acid position 636 of SEQ ID NO: 3 (P636L),a substitution of threonine (T) for the wild type residue isoleucine (I)at amino acid position 713 of SEQ ID NO: 3 (I713T), a substitution ofproline (P) for the wild type residue glutamine (Q) at amino acidposition 501 of SEQ ID NO: 5 (Q501P), a substitution of glutamine (Q)for the wild type residue lysine (K) at amino acid position 243 of SEQID NO: 3 (K243Q), a substitution of aspartic acid (D) for the wild typeresidue glutamic acid (E) at amino acid position 130 of SEQ ID NO: 5(E130D), a substitution of glycine (G) for the wild type residuearginine (R) at amino acid position 509 of SEQ ID NO: 3 (R509G), asubstitution of histidine (H) for the wild type residue arginine (R) atamino acid position 566 of SEQ ID NO: 3 (R566H), a substitution ofhistidine (H) for the wild type residue aspartic acid (D) at amino acidposition 677 of SEQ ID NO: 3 (D677H), a substitution of asparagine (N)for the wild type residue lysine (K) at amino acid position 466 of SEQID NO: 5 (K466N), a substitution of histidine (H) for the wild typeresidue arginine (R) at amino acid position 78 of SEQ ID NO: 3 (R78H), asubstitution of methionine (M) for the wild type residue lysine (K) atamino acid position 1 of SEQ ID NO: 6 (K6M), a substitution of leucine(L) for the wild type residue serine (S) at amino acid position 538 ofSEQ ID NO: 3 (S538L), a substitution of glutamine (Q) for the wild typeresidue leucine (L) at amino acid position 149 of SEQ ID NO: 3 (L149Q),a substitution of valine (V) for the wild type residue leucine (L) atamino acid position 252 of SEQ ID NO: 3 (L252V), a substitution ofvaline (V) for the wild type residue leucine (L) at amino acid position674 of SEQ ID NO: 3 (L674V), a substitution of valine (V) for the wildtype residue alanine (A) at amino acid position 656 of SEQ ID NO: 3(A656V), a substitution of aspartic acid (D) for the wild type residuealanine (A) at amino acid position 731 of SEQ ID NO: 3 (Y731D), asubstitution of threonine (T) for the wild type residue alanine (A) atamino acid position 345 of SEQ ID NO: 3 (A345T), a substitution ofaspartic acid (D) for the wild type residue alanine (A) at amino acidposition 244 of SEQ ID NO: 3 (Y244D), a substitution of tryptophan (W)for the wild type residue cysteine (C) at amino acid position 576 of SEQID NO: 3 (C576W), a substitution of lysine (K) for the wild type residueasparagine (N) at amino acid position 640 of SEQ ID NO: 3 (N640K), asubstitution of lysine (K) for the wild type residue asparagine (N) atamino acid position 675 of SEQ ID NO: 3 (N675K), a substitution oftyrosine (Y) for the wild type residue aspartic acid (D) at amino acidposition 579 of SEQ ID NO: 11 (D579Y), a substitution of isoleucine (I)for the wild type residue asparagine (N) at amino acid position 693 ofSEQ ID NO: 3 (N693I), and a substitution of lysine (K) for the wild typeresidue asparagine (N) at amino acid position 693 of SEQ ID NO: 3(N693K).

Other mutations of EZH2 can include: a frameshift at amino acid position730, 391, 461, 441, 235, 254, 564, 662, 715, 405, 685, 64, 73, 656, 718,374, 592, 505, 730, or 363 of SEQ ID NO: 3, 5 or 11, or thecorresponding nucleotide position of the nucleic acid sequence encodingSEQ ID NO: 3, 5, or 11; a deletion of glutamic acid (E) and leucine (L)at amino acid positions 148 and 149 of SEQ ID NO: 3, 5 or 11, or anonsense mutation at amino acid position 733, 25, 317, 62, 553, 328, 58,207, 123, 63, 137, or 60 of SEQ ID NO: 3, 5 or 11.

A subject of the present invention may have resistance to any one ormore other therapeutic agents or any of the compounds described herein.For example, the subject may have resistance to EZH inhibitors orprednisone.

The present invention features a method for inhibiting cancer cellproliferation comprising contacting said cancer cells with a compositioncomprising any compound of Formula (IIa) or pharmaceutically acceptablesalt thereof, and one or more additional therapeutic agent. Inhibitingcancer cell proliferation includes delaying cancer cell growth, inducingcell death, reducing cancer cell viability, inhibiting or delaying tumorgrowth, or reducing tumor size.

The present invention features methods of combination therapy whereinany compound of Formula (IIa), or pharmaceutically acceptable saltthereof, and one or more other therapeutic agents are administeredsimultaneously or sequentially. For example, any compound of Formula(IIa) or pharmaceutically acceptable salt thereof may be administeredprior to administration of one or more other therapeutic agents. Forexample, any compound of Formula (IIa) or pharmaceutically acceptablesalt there or may be administered prior to administration of acomposition comprising any compound of Formula (IIa) or pharmaceuticallyacceptable salt thereof and one or more other therapeutic agents.Concurrent or sequential administration of any compound of Formula (IIa)and/or each therapeutic agent can be effected by any appropriate routeincluding, but not limited to, oral routes, intravenous routes,intramuscular routes, and direct absorption through mucous membranetissues. The therapeutic agents can be administered by the same route orby different routes.

The methods of combination therapy featured in the present invention mayresult in a synergistic effect, wherein the effect of a combination ofcompounds or other therapeutic agents is greater than the sum of theeffects resulting from administration of any of the compounds or othertherapeutic agents as single agents. A synergistic effect may also be aneffect that cannot be achieved by administration of any of the compoundsor other therapeutic agents as single agents. The synergistic effect mayinclude, but is not limited to, an effect of treating cancer by reducingtumor size, inhibiting tumor growth, or increasing survival of thesubject. The synergistic effect may also include reducing cancer cellviability, inducing cancer cell death, and inhibiting or delaying cancercell growth.

Compositions of the present invention can be administered at a dosage of0.01 mg/kg per day to about 1000 mg/kg per day. Any compound of Formula(IIa) or pharmaceutically acceptable salt thereof may be administered ata dosage of 0.01 mg/kg per day to about 1000 mg/kg per day. Any othertherapeutic agent may be administered at a dosage of 0.01 mg/kg per dayto about 1000 mg/kg per day.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTIONS OF FIGURES

FIG. 1 is a schematic detailing in vitro treatment schedules fordetermining the effect of combination therapy on cell viability. (A)Administration of Compound 44 prior to administration of Compound 44 andcomponents of CHOP (Cyclophosphamide, Doxorubicin, Vincristine, andPrednisolone). (B) Administration of Compound 44 prior to CHOPcomponents. (C) Administration of components of CHOP prior toadministration of Compound 44 and CHOP components. After 4 days, cellviability was determined.

FIG. 2 is four graphs measuring cancer cell viability in vitro aftertreatment with Compound 44 and CHOP components alone or in combination.WSU-DLCL2 cells were treated as shown in FIG. 1A. Cells were firsttreated with varying concentrations of Compound 44. After four days,cells were treated with the combination of varying concentrations ofCompound 44 and (A) Mafofsamide (Cyclophosphamide metabolite), (B)Doxorubicin, (C) Vincristine, or (D) Prednisolone (Prednisonemetabolite). Control cells were treated with DMSO. Cell viability wasnormalized to the percentage of cell viability in DMSO-treated for eachCompound 44 concentration.

FIG. 3 is three graphs measuring cancer cell viability after varioustreatment schedules. WSU-DCLC2 cells were treated with increasingconcentrations of Prednisolone and Compound 44. Cells were treated withCompound 44 and Prednisolone according to the treatment schedule in (A)FIG. 1A, (B) FIG. 1B, or (C) FIG. 1C. Cell viability was normalized tothe DMSO/DMSO-treated sample.

FIG. 4 is three graphs measuring cancer cell viability after treatmentwith Prednisolone and Compound 44 in cell lines with different EZH2mutations. Cells were treated as depicted in FIG. 1A and with increasingconcentrations of Prednisolone and Compound 44. Cell viability wasnormalized to the DMSO/DMSO-treated sample. (A) WSU-DLCL2 cells expressthe Y641F mutation and are sensitive to EZH2 inhibitors. (B) RL cellsexpress the Y641N mutation are resistant to EZH2 inhibitors andPrednisolone. (C) OCI-LY19 cells are wild-type at Y641 and showsensitivity to Presnisolone, but do not show sensitivity to Compound 44or increased sensitivity to combinations of Compound 44 plusPrednisolone.

FIG. 5 is five graphs showing a panel of pharmacokinetic profiles forco-administration of Compound 44 and CHOP components in vivo. BALB/cmice were administered a single oral administration of Compound 44 andCHOP components. The concentration of Compound 44 (ng/mL) in the plasmawas measured at various timepoints 0-24 hours after administration.Cyclophoshamide was administered by intraperitoneal injection at 30mg/kg. Vincristine was administered by intravenous injection at 0.375mg/kg. Doxorubicin was administered by intravenous injection at 2.475mg/kg. Prednisolone was administered by oral administration at 0.15mg/kg. Compound 44 was administered by oral administration at 225 mg/kg.

FIG. 6 is two graphs demonstrating the effect of Compound 44 and CHOPadministration in SUDHL6 xenograft model on tumor growth and survival.Athymic nude mice were subcutaneously injected with 1×10⁷ SUDHL6 humanlymphoma cells. After tumors reached a size of app. 120 mm³ Compound 44was administered over 28 days at the indicated dosages either threetimes a day (TID) or twice a day (BID). CHOP (Cyclophosphamide,Vincristine, Doxorubicin, and Prednisone) was administered at day 1 and8 to mice receiving 225 mg/kg twice per day (225 mg/kg BID and CHOP).Control mice did not receive any Compound 44 (vehicle) or CHOP only(CHOP). Tumor volume was measured twice a week. Animals were euthanizedwhen tumor volume reached 2000 mm³ or 60 days after the first dose. (A)For tumor growth delay analysis, median tumor volume was calculated foreach treatment group by measuring the tumors twice a week for 60 days oruntil the tumor reached 2000 mm³. (B) Kaplan-meier curve depicts thesurvival rate of the mice.

FIG. 7 is three graphs demonstrating the effect of Compound 44 and CHOPadministration in WSU-DLCL2 xenograft model on tumor growth andsurvival. SCID mice were subcutaneously injected with 1×10⁷ WSU-DLCL2human lymphoma cells. After tumors reached a size of app. 120 mm³Compound 44 was administered over 28 days at the indicated dosages atthree times a day (TID), twice a day (BID) or once a day (QD). CHOP(Cyclophosphamide, Vincristine, Doxorubicin, and Prednisone) wasadministered at day 1 and 22 to mice receiving 225 mg/kg twice a day(225 mg/kg BID and CHOP). Control mice either did not receive anyCompound 44 (vehicle) or received CHOP only (CHOP). Tumor volume wasmeasured twice a week. Animals were euthanized 28 days after first dose.(A) Treatment efficacy was determined by measuring mean tumor volumeover the course of treatment. (B) Concentration (ng/mL) of Compound 44was measured in plasma from mice on day 28 at before administration(trough) and three hours post-administration (post). (C) Concentration(ng/g) of Compound 44 was measured from tumor tissue from mice on day 28three hours post-administration.

FIG. 8 is a western blot analysis and a graph showing the histonemethylation status from tumor samples from the WSU-DLCL2 xenograftmodel. Tumors were harvested 28 days after injection and histones wereextracted. (A) A Western Blot was probed with antibodies specificallyrecognizing tri-methylated Lysine 27 of histone H3 (H3K27me3) and totalhistone H3 proteins. (B) Methylation status of tumors was determined byELISA. H3K27 tri-methylation was detected and normalized to totalhistone H3 levels.

FIG. 9 is four graphs demonstrating the effect of Compound 44 and COPadministration in a SUDHL10 xenograft model on tumor growth andsurvival. SCID mice were subcutaneously injected with 1×10⁷ SUDHL10human lymphoma cells. After tumors reached a size of app. 120 mm³Compound 44 was administered over 28 days at the indicated dosages twicea day (BID). COP (Cyclophosphamide, Vincristine, and Prednisone) wasadministered at day 1 and 22 to mice receiving 250 mg/kg twice a day(250 mg/kg BID and COP). Control mice did not receive any Compound 44(vehicle) or COP only (COP). Tumor volume was measured twice a week. (A)Treatment efficacy was determined by measuring mean tumor volume overthe course of treatment. (B) Mean tumor weight was determined after micewere euthanized on day 28 after first dosing. (C) For tumor growth delayanalysis, mean tumor volume and SEM was calculated for each treatmentgroup by measuring the tumors twice a week for 60 days or until thetumor reached 2000 mm³. (D) Kaplan-Meier curve depicts the survival rateof the treated mice.

FIG. 10 is four graphs showing the pharmacokinetic and pharmacodynamicprofiles after COP and Compound 44 administration in the SUDHL10xenograft model. Tumor-bearing mice were euthanized after 28 days oftreatment and tissues were harvested. (A) Concentration (ng/mL) ofCompound 44 was measured in plasma from mice on day 28 beforeadministration (trough) and post-administration (post) by LC-MS/MS.Methylation status of various tissues from tumor-bearing mice weredetermined by ELISA: (B) tumor, (C) spleen, and (D) bone marrow. H3K27tri-methylation was detected and normalized to total histone H3 levels.

FIG. 11 is a structure model of partial EZH2 protein based on the Achain of nuclear receptor binding SET domain protein 1 (NSD1) isprovided below. This model corresponds to amino acid residues 533-732 ofEZH2 sequence of SEQ ID NO: 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the discovery that EZH2 histonemethyltransferase inhibitors and other anti-cancer agents can be used incombination to treat certain tumors with superior results than thoseachieved by treating tumors with EZH2 histone methyltransferaseinhibitors and the anti-cancer agents alone. Accordingly, the presentinvention provides a composition comprising an EZH2 histonemethyltransferase inhibitor and one or more other therapeutic agents,and methods for their use to treat diseases the course of which can beinfluenced by modulating the methylation status of histones or otherproteins, e.g., cancer. In a certain embodiment, the present inventionfeatures a composition comprising a compound of Formula (IIa) andprednisone. The present invention also includes methods for combinationtherapies comprising EZH2 histone methyltransferase inhibitor and one ormore therapeutic agents, such as a compound of Formula (IIa) andprednisone, to treat cancer, e.g., follicular lymphoma (FL) and diffusecell large B-cell lymphoma (DCLBL). Specifically, the methods of thepresent invention are useful for treating or preventing cancer orinhibiting cancer cell proliferation.

EZH2 is a histone methyltransferase that is the catalytic subunit of thePRC2 complex which catalyzes the mono-through tri-methylation of lysine27 on histone H3 (H3-K27). Histone H3-K27 trimethylation is a mechanismfor suppressing transcription of specific genes that are proximal to thesite of histone modification. This trimethylation is known to be acancer marker with altered expression in cancer, such as prostate cancer(see, e.g., U.S. Patent Application Publication No. 2003/0175736;incorporated herein by reference in its entirety). Other studiesprovided evidence for a functional link between dysregulated EZH2expression, transcriptional repression, and neoplastic transformation.Varambally et al. (2002) Nature 419(6907):624-9 Kleer et al. (2003) ProcNatlAcad Sci USA 100(20):11606-11.

An aspect of the present invention relates to methods for treating oralleviating a symptom of cancer or precancerous condition in a subjectby administering to a subject expressing a mutant EZH2 a therapeuticallyeffective amount of an EZH2 inhibitor and one or more other therapeuticagents. The mutant EZH2 of the present invention refers to a mutant EZH2polypeptide or a nucleic acid sequence encoding a mutant EZH2polypeptide. In certain embodiments the mutant EZH2 comprises one ormore mutations in its substrate pocket domain as defined in SEQ ID NO:6. For example, the mutation may be a substitution, a point mutation, anonsense mutation, a misssense mutation, a deletion, or an insertion.

Human EZH2 nucleic acids and polypeptides have previously beendescribed. See, e.g., Chen et al. (1996) Genomics 38:30-7 [746 aminoacids]; Swiss-Prot Accession No. Q15910 [746 amino acids]; GenBankAccession Nos. NM_004456 and NP_004447 (isoform a [751 amino acids]);and GenBank Accession Nos. NM_152998 and NP_694543 (isoform b

Amino acid sequence of human EZH2 (Swiss-Prot Accession No.Q15910) (SEQ ID NO: 1)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGIKTCRQVYEERVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPNucleotide sequence of human EZH2, transcript variant 1(GenBank Accession No. NM_004456) (SEQ ID NO: 2)ggcggcgcttgattgggctgggggggccaaataaaagcgatggcgattgggctgccgcgtttggcgctcggtccggtcgcgtccgacacccggtgggactcagaaggcagtggagccccggcggcggcggcggcggcgcgcgggggcgacgcgcgggaacaacgcgagtcggcgcgcgggacgaagaataatcatgggccagactgggaagaaatctgagaagggaccagtttgttggcggaagcgtgtaaaatcagagtacatgcgactgagacagctcaagaggttcagacgagctgatgaagtaaagagtatgtttagttccaatcgtcagaaaattttggaaagaacggaaatcttaaaccaagaatggaaacagcgaaggatacagcctgtgcacatcctgacttctgtgagctcattgcgcgggactagggagtgttcggtgaccagtgacttggattttccaacacaagtcatcccattaaagactctgaatgcagttgcttcagtacccataatgtattcttggtctcccctacagcagaattttatggtggaagatgaaactgttttacataacattccttatatgggagatgaagttttagatcaggatggtactttcattgaagaactaataaaaaattatgatgggaaagtacacggggatagagaatgtgggtttataaatgatgaaatttttgtggagttggtgaatgcccttggtcaatataatgatgatgacgatgatgatgatggagacgatcctgaagaaagagaagaaaagcagaaagatctggaggatcaccgagatgataaagaaagccgcccacctcggaaatttccttctgataaaatttttgaagccatttcctcaatgtttccagataagggcacagcagaagaactaaaggaaaaatataaagaactcaccgaacagcagctcccaggcgcacttcctcctgaatgtacccccaacatagatggaccaaatgctaaatctgttcagagagagcaaagcttacactcctttcatacgcttttctgtaggcgatgttttaaatatgactgcttcctacatcgtaagtgcaattattcttttcatgcaacacccaacacttataagcggaagaacacagaaacagctctagacaacaaaccttgtggaccacagtgttaccagcatttggagggagcaaaggagtttgctgctgctctcaccgctgagcggataaagaccccaccaaaacgtccaggaggccgcagaagaggacggcttcccaataacagtagcaggcccagcacccccaccattaatgtgctggaatcaaaggatacagacagtgatagggaagcagggactgaaacggggggagagaacaatgataaagaagaagaagagaagaaagatgaaacttcgagctcctctgaagcaaattctcggtgtcaaacaccaataaagatgaagccaaatattgaacctcctgagaatgtggagtggagtggtgctgaagcctcaatgtttagagtcctcattggcacttactatgacaatttctgtgccattgctaggttaattgggaccaaaacatgtagacaggtgtatgagtttagagtcaaagaatctagcatcatagctccagctcccgctgaggatgtggatactcctccaaggaaaaagaagaggaaacaccggttgtgggctgcacactgcagaaagatacagctgaaaaaggacggctcctctaaccatgtttacaactatcaaccctgtgatcatccacggcagccttgtgacagttcgtgcccttgtgtgatagcacaaaatttttgtgaaaagttttgtcaatgtagttcagagtgtcaaaaccgctttccgggatgccgctgcaaagcacagtgcaacaccaagcagtgcccgtgctacctggctgtccgagagtgtgaccctgacctctgtcttacttgtggagccgctgaccattgggacagtaaaaatgtgtcctgcaagaactgcagtattcagcggggctccaaaaagcatctattgctggcaccatctgacgtggcaggctgggggatttttatcaaagatcctgtgcagaaaaatgaattcatctcagaatactgtggagagattatttctcaagatgaagctgacagaagagggaaagtgtatgataaatacatgtgcagctttctgttcaacttgaacaatgattttgtggtggatgcaacccgcaagggtaacaaaattcgttttgcaaatcattcggtaaatccaaactgctatgcaaaagttatgatggttaacggtgatcacaggataggtatttttgccaagagagccatccagactggcgaagagctgttttttgattacagatacagccaggctgatgccctgaagtatgtcggcatcgaaagagaaatggaaatcccttgacatctgctacctcctcccccctcctctgaaacagctgccttagcttcaggaacctcgagtactgtgggcaatttagaaaaagaacatgcagtttgaaattctgaatttgcaaagtactgtaagaataatttatagtaatgagtttaaaaatcaactttttattgccttctcaccagctgcaaagtgttttgtaccagtgaatttttgcaataatgcagtatggtacatttttcaactttgaataaagaatacttgaacttgtccttgttgaatcFull amino acid of EZH2, isoform a (GenBank Accession No.NP_004447) (SEQ ID NO: 3)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHRKCNYSFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDIDSDREAGTEIGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPNucleotide sequence of human EZH2, transcript variant 2(GenBank Accession No. NM_152998) (SEQ ID NO: 4)ggcggcgcttgattgggctgggggggccaaataaaagcgatggcgattgggctgccgcgtttggcgctcggtccggtcgcgtccgacacccggtgggactcagaaggcagtggagccccggcggcggcggcggcggcgcgcgggggcgacgcgcgggaacaacgcgagtcggcgcgcgggacgaagaataatcatgggccagactgggaagaaatctgagaagggaccagtttgttggcggaagcgtgtaaaatcagagtacatgcgactgagacagctcaagaggttcagacgagctgatgaagtaaagagtatgtttagttccaatcgtcagaaaattttggaaagaacggaaatcttaaaccaagaatggaaacagcgaaggatacagcctgtgcacatcctgacttctgtgagctcattgcgcgggactagggaggtggaagatgaaactgttttacataacattccttatatgggagatgaagttttagatcaggatggtactttcattgaagaactaataaaaaattatgatgggaaagtacacggggatagagaatgtgggtttataaatgatgaaatttttgtggagttggtgaatgcccttggtcaatataatgatgatgacgatgatgatgatggagacgatcctgaagaaagagaagaaaagcagaaagatctggaggatcaccgagatgataaagaaagccgcccacctcggaaatttccttctgataaaatttttgaagccatttcctcaatgtttccagataagggcacagcagaagaactaaaggaaaaatataaagaactcaccgaacagcagctcccaggcgcacttcctcctgaatgtacccccaacatagatggaccaaatgctaaatctgttcagagagagcaaagcttacactcctttcatacgcttttctgtaggcgatgttttaaatatgactgcttcctacatccttttcatgcaacacccaacacttataagcggaagaacacagaaacagctctagacaacaaaccttgtggaccacagtgttaccagcatttggagggagcaaaggagtttgctgctgctctcaccgctgagcggataaagaccccaccaaaacgtccaggaggccgcagaagaggacggcttcccaataacagtagcaggcccagcacccccaccattaatgtgctggaatcaaaggatacagacagtgatagggaagcagggactgaaacggggggagagaacaatgataaagaagaagaagagaagaaagatgaaacttcgagctcctctgaagcaaattctcggtgtcaaacaccaataaagatgaagccaaatattgaacctcctgagaatgtggagtggagtggtgctgaagcctcaatgtttagagtcctcattggcacttactatgacaatttctgtgccattgctaggttaattgggaccaaaacatgtagacaggtgtatgagtttagagtcaaagaatctagcatcatagctccagctcccgctgaggatgtggatactcctccaaggaaaaagaagaggaaacaccggttgtgggctgcacactgcagaaagatacagctgaaaaaggacggctcctctaaccatgtttacaactatcaaccctgtgatcatccacggcagccttgtgacagttcgtgcccttgtgtgatagcacaaaatttttgtgaaaagttttgtcaatgtagttcagagtgtcaaaaccgctttccgggatgccgctgcaaagcacagtgcaacaccaagcagtgcccgtgctacctggctgtccgagagtgtgaccctgacctctgtcttacttgtggagccgctgaccattgggacagtaaaaatgtgtcctgcaagaactgcagtattcagcggggctccaaaaagcatctattgctggcaccatctgacgtggcaggctgggggatttttatcaaagatcctgtgcagaaaaatgaattcatctcagaatactgtggagagattatttctcaagatgaagctgacagaagagggaaagtgtatgataaatacatgtgcagctttctgttcaacttgaacaatgattttgtggtggatgcaacccgcaagggtaacaaaattcgttttgcaaatcattcggtaaatccaaactgctatgcaaaagttatgatggttaacggtgatcacaggataggtatttttgccaagagagccatccagactggcgaagagctgttttttgattacagatacagccaggctgatgccctgaagtatgtcggcatcgaaagagaaatggaaatcccttgacatctgctacctcctcccccctcctctgaaacagctgccttagcttcaggaacctcgagtactgtgggcaatttagaaaaagaacatgcagtttgaaattctgaatttgcaaagtactgtaagaataatttatagtaatgagtttaaaaatcaactttttattgccttctcaccagctgcaaagtgttttgtaccagtgaatttttgcaataatgcagtatggtacatttttcaactttgaataaagaatacttgaacttgtc cttgttgaatcFull amino acid of EZH2, isoform b (GenBank Accession No. NP_694543)(SEQ ID NO: 5) MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTREVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGIKTCRQVYEERVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGI EREMEIPFull amino acid of EZH2, isoform e (GenBank Accession No.NP_001190178.1) (SEQ ID NO: 11)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSCSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNECATARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKGQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPHomo sapiens enhancer of zeste homolog 2 (Drosophila) (EZH2),transcript variant 5, nucleotide sequence (GenBank Accession No.NM_001203249.1) (SEQ ID NO: 12)GACGACGTTCGCGGCGGGGAACTCGGAGTAGCTTCGCCTCTGACGTTTCCCCACGACGCACCCCGAAATCCCCCTGAGCTCCGGCGGTCGCGGGCTGCCCTCGCCGCCTGGTCTGGCTTTATGCTAAGTTTGAGGGAAGAGTCGAGCTGCTCTGCTCTCTATTGATTGTGTTTCTGGAGGGCGTCCTGTTGAATTCCCACTTCATTGTGTACATCCCCTTCCGTTCCCCCCAAAAATCTGTGCCACAGGGTTACTTTTTGAAAGCGGGAGGAATCGAGAAGCACGATCTTTTGGAAAACTTGGTGAACGCCTAAATAATCATGGGCCAGACTGGGAAGAAATCTGAGAAGGGACCAGTTTGTTGGCGGAAGCGTGTAAAATCAGAGTACATGCGACTGAGACAGCTCAAGAGGTTCAGACGAGCTGATGAAGTAAAGAGTATGTTTAGTTCCAATCGTCAGAAAATTTTGGAAAGAACGGAAATCTTAAACCAAGAATGGAAACAGCGAAGGATACAGCCTGTGCACATCCTGACTTCTTGTTCGGTGACCAGTGACTTGGATTTTCCAACACAAGTCATCCCATTAAAGACTCTGAATGCAGTTGCTTCAGTACCCATAATGTATTCTTGGTCTCCCCTACAGCAGAATTTTATGGTGGAAGATGAAACTGTTTTACATAACATTCCTTATATGGGAGATGAAGTTTTAGATCAGGATGGTACTTTCATTGAAGAACTAATAAAAAATTATGATGGGAAAGTACACGGGGATAGAGAATGTGGGTTTATAAATGATGAAATTTTTGTGGAGTTGGTGAATGCCCTTGGTCAATATAATGATGATGACGATGATGATGATGGAGACGATCCTGAAGAAAGAGAAGAAAAGCAGAAAGATCTGGAGGATCACCGAGATGATAAAGAAAGCCGCCCACCTCGGAAATTTCCTTCTGATAAAATTTTTGAAGCCATTTCCTCAATGTTTCCAGATAAGGGCACAGCAGAAGAACTAAAGGAAAAATATAAAGAACTCACCGAACAGCAGCTCCCAGGCGCACTTCCTCCTGAATGTACCCCCAACATAGATGGACCAAATGCTAAATCTGTTCAGAGAGAGCAAAGCTTACACTCCTTTCATACGCTTTTCTGTAGGCGATGTTTTAAATATGACTGCTTCCTACATCCTTTTCATGCAACACCCAACACTTATAAGCGGAAGAACACAGAAACAGCTCTAGACAACAAACCTTGTGGACCACAGTGTTACCAGCATTTGGAGGGAGCAAAGGAGTTTGCTGCTGCTCTCACCGCTGAGCGGATAAAGACCCCACCAAAACGTCCAGGAGGCCGCAGAAGAGGACGGCTTCCCAATAACAGTAGCAGGCCCAGCACCCCCACCATTAATGTGCTGGAATCAAAGGATACAGACAGTGATAGGGAAGCAGGGACTGAAACGGGGGGAGAGAACAATGATAAAGAAGAAGAAGAGAAGAAAGATGAAACTTCGAGCTCCTCTGAAGCAAATTCTCGGTGTCAAACACCAATAAAGATGAAGCCAAATATTGAACCTCCTGAGAATGTGGAGTGGAGTGGTGCTGAAGCCTCAATGTTTAGAGTCCTCATTGGCACTTACTATGACAATTTCTGTGCCATTGCTAGGTTAATTGGGACCAAAACATGTAGACAGGTGTATGAGTTTAGAGTCAAAGAATCTAGCATCATAGCTCCAGCTCCCGCTGAGGATGTGGATACTCCTCCAAGGAAAAAGAAGAGGAAACACCGGTTGTGGGCTGCACACTGCAGAAAGATACAGCTGAAAAAGGGTCAAAACCGCTTTCCGGGATGCCGCTGCAAAGCACAGTGCAACACCAAGCAGTGCCCGTGCTACCTGGCTGTCCGAGAGTGTGACCCTGACCTCTGTCTTACTTGTGGAGCCGCTGACCATTGGGACAGTAAAAATGTGTCCTGCAAGAACTGCAGTATTCAGCGGGGCTCCAAAAAGCATCTATTGCTGGCACCATCTGACGTGGCAGGCTGGGGGATTTTTATCAAAGATCCTGTGCAGAAAAATGAATTCATCTCAGAATACTGTGGAGAGATTATTTCTCAAGATGAAGCTGACAGAAGAGGGAAAGTGTATGATAAATACATGTGCAGCTTTCTGTTCAACTTGAACAATGATTTTGTGGTGGATGCAACCCGCAAGGGTAACAAAATTCGTTTTGCAAATCATTCGGTAAATCCAAACTGCTATGCAAAAGTTATGATGGTTAACGGTGATCACAGGATAGGTATTTTTGCCAAGAGAGCCATCCAGACTGGCGAAGAGCTGTTTTTTGATTACAGATACAGCCAGGCTGATGCCCTGAAGTATGTCGGCATCGAAAGAGAAATGGAAATCCCTTGACATCTGCTACCTCCTCCCCCCTCCTCTGAAACAGCTGCCTTAGCTTCAGGAACCTCGAGTACTGTGGGCAATTTAGAAAAAGAACATGCAGTTTGAAATTCTGAATTTGCAAAGTACTGTAAGAATAATTTATAGTAATGAGTTTAAAAATCAACTTTTTATTGCCTTCTCACCAGCTGCAAAGTGTTTTGTACCAGTGAATTTTTGCAATAATGCAGTATGGTACATTTTTCAACTTTGAATAAAGAATACTTGAACTTGTCCTTGTTGAATC

A structure model of partial EZH2 protein based on the A chain ofnuclear receptor binding SET domain protein 1 (NSD1) is provided in FIG.11. This model corresponds to amino acid residues 533-732 of EZH2sequence of SEQ ID NO: 1.

The corresponding amino acid sequence of the structure model seen inFIG. 11 is provided below. The residues in the substrate pocket domainare underlined. The residues in the SET domain are shown italic.

(SEQ ID NO: 6)

The catalytic site of EZH2 is believed to reside in a conserved domainof the protein known as the SET domain. The amino acid sequence of theSET domain of EZH2 is provided by the following partial sequencespanning amino acid residues 613-726 of Swiss-Prot Accession No. Q15910(SEQ ID NO: 1):

(SEQ ID NO: 7) HLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFA KRAIQTGEELFFDY.The tyrosine (Y) residue shown underlined in SEQ ID NO: 7 is Tyr641(Y641) in Swiss-Prot Accession No. Q15910 (SEQ ID NO: 1).

The SET domain of GenBank Accession No. NP_004447 (SEQ ID NO: 3) spansamino acid residues 618-731 and is identical to SEQ ID NO:6. Thetyrosine residue corresponding to Y641 in Swiss-Prot Accession No.Q15910 shown underlined in SEQ ID NO: 7 is Tyr646 (Y646) in GenBankAccession No. NP_004447 (SEQ ID NO: 3).

The SET domain of GenBank Accession No. NP_694543 (SEQ ID NO: 5) spansamino acid residues 574-687 and is identical to SEQ ID NO: 7. Thetyrosine residue corresponding to Y641 in Swiss-Prot Accession No.Q15910 shown underlined in SEQ ID NO: 7 is Tyr602 (Y602) in GenBankAccession No. NP_694543 (SEQ ID NO: 5).

The nucleotide sequence encoding the SET domain of GenBank Accession No.NP_004447 is

(SEQ ID NO: 8) catctattgctggcaccatctgacgtggcaggctgggggatttttatcaaagatcctgtgcagaaaaatgaattcatctcagaatactgtggagagattatttctcaagatgaagctgacagaagagggaaagtgtatgataaatacatgtgcagctttctgttcaacttgaacaatgattttgtggtggatgcaacccgcaagggtaacaaaattcgttttgcaaatcattcggtaaatccaaactgctatgcaaaagttatgatggttaacggtgatcacaggataggtatttttgccaagagagccatccagactggcgaagagctgttttttgattac,where the codon encoding Y641 is shown underlined.

For purposes of this application, amino acid residue Y641 of human EZH2is to be understood to refer to the tyrosine residue that is orcorresponds to Y641 in Swiss-Prot Accession No. Q15910.

Full amino acid sequence of Y641 mutant EZH2 (SEQ ID NO: 9)MGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQKILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKIPPKRPGGRRRGRLPNNSSRPSTPTINVLESKDIDSDREAGTEIGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLIGTKICRQVYEFRVKESSIIAPARAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCNTKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEXCGEIISQDEADRRGKVYDKYMCSFLENLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPWherein x can be any amino acid residue other than tyrosine (Y)

Also for purposes of this application, a Y641 mutant of human EZH2, and,equivalently, a Y641 mutant of EZH2, is to be understood to refer to ahuman EZH2 in which the amino acid residue corresponding to Y641 ofwild-type human EZH2 is substituted by an amino acid residue other thantyrosine.

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of a single amino acid residue corresponding to Y641 ofwild-type human EZH2 by an amino acid residue other than tyrosine.

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of phenylalanine (F) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641F mutant or,equivalently, Y641F.

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of histidine (H) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641H mutant or,equivalently, Y641H.

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of asparagine (N) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641N mutant or,equivalently, Y641N.

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of serine (S) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641S mutant or,equivalently, Y641 S.

In one embodiment the amino acid sequence of a Y641 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of cysteine (C) for the single amino acid residuecorresponding to Y641 of wild-type human EZH2. The Y641 mutant of EZH2according to this embodiment is referred to herein as a Y641C mutant or,equivalently, Y641C.

In one embodiment the amino acid sequence of a A677 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of a non-alanine amino acid, preferably glycine (G) for thesingle amino acid residue corresponding to A677 of wild-type human EZH2.The A677 mutant of EZH2 according to this embodiment is referred toherein as an A677 mutant, and preferably an A677G mutant or,equivalently, A677G.

In one embodiment the amino acid sequence of a A687 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of a non-alanine amino acid, preferably valine (V) for thesingle amino acid residue corresponding to A687 of wild-type human EZH2.The A687 mutant of EZH2 according to this embodiment is referred toherein as an A687 mutant and preferably an A687V mutant or,equivalently, A687V.

In one embodiment the amino acid sequence of a R685 mutant of EZH2differs from the amino acid sequence of wild-type human EZH2 only bysubstitution of a non-arginine amino acid, preferably histidine (H) orcysteine (C) for the single amino acid residue corresponding to R685 ofwild-type human EZH2. The R685 mutant of EZH2 according to thisembodiment is referred to herein as an R685 mutant and preferably anR685C mutant or an R685H mutant or, equivalently, R685H or R685C.

In one embodiment the amino acid sequence of a mutant of EZH2 differsfrom the amino acid sequence of wild-type human EZH2 in one or moreamino acid residues in its substrate pocket domain as defined in SEQ IDNO: 6. The mutant of EZH2 according to this embodiment is referred toherein as an EZH2 mutant.

Other exemplary substitution amino acid mutation includes a substitutionat amino acid position 677, 687, 674, 685, or 641 of SEQ ID NO: 1, suchas, but is not limited to a substitution of glycine (G) for the wildtype residue alanine (A) at amino acid position 677 of SEQ ID NO: 1(A677G); a substitution of valine (V) for the wild type residue alanine(A) at amino acid position 687 of SEQ ID NO: 1 (A687V); a substitutionof methionine (M) for the wild type residue valine (V) at amino acidposition 674 of SEQ ID NO: 1 (V674M); a substitution of histidine (H)for the wild type residue arginine (R) at amino acid position 685 of SEQID NO: 1 (R685H); a substitution of cysteine (C) for the wild typeresidue arginine (R) at amino acid position 685 of SEQ ID NO: 1 (R685C);a substitution of phenylalanine (F) for the wild type residue tyrosine(Y) at amino acid position 641 of SEQ ID NO: 1 (Y641F); a substitutionof histidine (H) for the wild type residue tyrosine (Y) at amino acidposition 641 of SEQ ID NO: 1 (Y641H); a substitution of asparagine (N)for the wild type residue tyrosine (Y) at amino acid position 641 of SEQID NO: 1 (Y641N); a substitution of serine (S) for the wild type residuetyrosine (Y) at amino acid position 641 of SEQ ID NO: 1 (Y641S); or asubstitution of cysteine (C) for the wild type residue tyrosine (Y) atamino acid position 641 of SEQ ID NO: 1 (Y641C).

The mutation of the present invention may also include a substitution ofserine (S) for the wild type residue asparagine (N) at amino acidposition 322 of SEQ ID NO: 3 (N322S), a substitution of glutamine (Q)for the wild type residue arginine (R) at amino acid position 288 of SEQID NO: 3 (R288Q), a substitution of isoleucine (I) for the wild typeresidue threonine (T) at amino acid position 573 of SEQ ID NO: 3(T5731), a substitution of glutamic acid (E) for the wild type residueaspartic acid (D) at amino acid position 664 of SEQ ID NO: 3 (D664E), asubstitution of glutamine (Q) for the wild type residue arginine (R) atamino acid position 458 of SEQ ID NO: 5 (R458Q), a substitution oflysine (K) for the wild type residue glutamic acid (E) at amino acidposition 249 of SEQ ID NO: 3 (E249K), a substitution of cysteine (C) forthe wild type residue arginine (R) at amino acid position 684 of SEQ IDNO: 3 (R684C), a substitution of histidine (H) for the wild type residuearginine (R) at amino acid position 628 of SEQ ID NO: 11 (R628H), asubstitution of histidine (H) for the wild type residue glutamine (Q) atamino acid position 501 of SEQ ID NO: 5 (Q501H), a substitution ofasparagine (N) for the wild type residue aspartic acid (D) at amino acidposition 192 of SEQ ID NO: 3 (D192N), a substitution of valine (V) forthe wild type residue aspartic acid (D) at amino acid position 664 ofSEQ ID NO: 3 (D664V), a substitution of leucine (L) for the wild typeresidue valine (V) at amino acid position 704 of SEQ ID NO: 3 (V704L), asubstitution of serine (S) for the wild type residue proline (P) atamino acid position 132 of SEQ ID NO: 3 (P132S), a substitution oflysine (K) for the wild type residue glutamic acid (E) at amino acidposition 669 of SEQ ID NO: 11 (E669K), a substitution of threonine (T)for the wild type residue alanine (A) at amino acid position 255 of SEQID NO: 3 (A255T), a substitution of valine (V) for the wild type residueglutamic acid (E) at amino acid position 726 of SEQ ID NO: 3 (E726V), asubstitution of tyrosine (Y) for the wild type residue cysteine (C) atamino acid position 571 of SEQ ID NO: 3 (C571Y), a substitution ofcysteine (C) for the wild type residue phenylalanine (F) at amino acidposition 145 of SEQ ID NO: 3 (F145C), a substitution of threonine (T)for the wild type residue asparagine (N) at amino acid position 693 ofSEQ ID NO: 3 (N693T), a substitution of serine (S) for the wild typeresidue phenylalanine (F) at amino acid position 145 of SEQ ID NO: 3(F145S), a substitution of histidine (H) for the wild type residueglutamine (Q) at amino acid position 109 of SEQ ID NO: 11 (Q109H), asubstitution of cysteine (C) for the wild type residue phenylalanine (F)at amino acid position 622 of SEQ ID NO: 11 (F622C), a substitution ofarginine (R) for the wild type residue glycine (G) at amino acidposition 135 of SEQ ID NO: 3 (G135R), a substitution of glutamine (Q)for the wild type residue arginine (R) at amino acid position 168 of SEQID NO: 5 (R168Q), a substitution of arginine (R) for the wild typeresidue glycine (G) at amino acid position 159 of SEQ ID NO: 3 (G159R),a substitution of cysteine (C) for the wild type residue arginine (R) atamino acid position 310 of SEQ ID NO: 5 (R310C), a substitution ofhistidine (H) for the wild type residue arginine (R) at amino acidposition 561 of SEQ ID NO: 3 (R561H), a substitution of histidine (H)for the wild type residue arginine (R) at amino acid position 634 of SEQID NO: 11 (R634H), a substitution of arginine (R) for the wild typeresidue glycine (G) at amino acid position 660 of SEQ ID NO: 3 (G660R),a substitution of cysteine (C) for the wild type residue tyrosine (Y) atamino acid position 181 of SEQ ID NO: 3 (Y181C), a substitution ofarginine (R) for the wild type residue histidine (H) at amino acidposition 297 of SEQ ID NO: 3 (H297R), a substitution of serine (S) forthe wild type residue cysteine (C) at amino acid position 612 of SEQ IDNO: 11 (C612S), a substitution of tyrosine (Y) for the wild type residuehistidine (H) at amino acid position 694 of SEQ ID NO: 3 (H694Y), asubstitution of alanine (A) for the wild type residue aspartic acid (D)at amino acid position 664 of SEQ ID NO: 3 (D664A), a substitution ofthreonine (T) for the wild type residue isoleucine (I) at amino acidposition 150 of SEQ ID NO: 3 (I150T), a substitution of arginine (R) forthe wild type residue isoleucine (I) at amino acid position 264 of SEQID NO: 3 (I264R), a substitution of leucine (L) for the wild typeresidue proline (P) at amino acid position 636 of SEQ ID NO: 3 (P636L),a substitution of threonine (T) for the wild type residue isoleucine (I)at amino acid position 713 of SEQ ID NO: 3 (I713T), a substitution ofproline (P) for the wild type residue glutamine (Q) at amino acidposition 501 of SEQ ID NO: 5 (Q501P), a substitution of glutamine (Q)for the wild type residue lysine (K) at amino acid position 243 of SEQID NO: 3 (K243Q), a substitution of aspartic acid (D) for the wild typeresidue glutamic acid (E) at amino acid position 130 of SEQ ID NO: 5(E130D), a substitution of glycine (G) for the wild type residuearginine (R) at amino acid position 509 of SEQ ID NO: 3 (R509G), asubstitution of histidine (H) for the wild type residue arginine (R) atamino acid position 566 of SEQ ID NO: 3 (R566H), a substitution ofhistidine (H) for the wild type residue aspartic acid (D) at amino acidposition 677 of SEQ ID NO: 3 (D677H), a substitution of asparagine (N)for the wild type residue lysine (K) at amino acid position 466 of SEQID NO: 5 (K466N), a substitution of histidine (H) for the wild typeresidue arginine (R) at amino acid position 78 of SEQ ID NO: 3 (R78H), asubstitution of methionine (M) for the wild type residue lysine (K) atamino acid position 1 of SEQ ID NO: 6 (K6M), a substitution of leucine(L) for the wild type residue serine (S) at amino acid position 538 ofSEQ ID NO: 3 (S538L), a substitution of glutamine (Q) for the wild typeresidue leucine (L) at amino acid position 149 of SEQ ID NO: 3 (L149Q),a substitution of valine (V) for the wild type residue leucine (L) atamino acid position 252 of SEQ ID NO: 3 (L252V), a substitution ofvaline (V) for the wild type residue leucine (L) at amino acid position674 of SEQ ID NO: 3 (L674V), a substitution of valine (V) for the wildtype residue alanine (A) at amino acid position 656 of SEQ ID NO: 3(A656V), a substitution of aspartic acid (D) for the wild type residuealanine (A) at amino acid position 731 of SEQ ID NO: 3 (Y731D), asubstitution of threonine (T) for the wild type residue alanine (A) atamino acid position 345 of SEQ ID NO: 3 (A345T), a substitution ofaspartic acid (D) for the wild type residue alanine (A) at amino acidposition 244 of SEQ ID NO: 3 (Y244D), a substitution of tryptophan (W)for the wild type residue cysteine (C) at amino acid position 576 of SEQID NO: 3 (C576W), a substitution of lysine (K) for the wild type residueasparagine (N) at amino acid position 640 of SEQ ID NO: 3 (N640K), asubstitution of lysine (K) for the wild type residue asparagine (N) atamino acid position 675 of SEQ ID NO: 3 (N675K), a substitution oftyrosine (Y) for the wild type residue aspartic acid (D) at amino acidposition 579 of SEQ ID NO: 11 (D579Y), a substitution of isoleucine (I)for the wild type residue asparagine (N) at amino acid position 693 ofSEQ ID NO: 3 (N693I), and a substitution of lysine (K) for the wild typeresidue asparagine (N) at amino acid position 693 of SEQ ID NO: 3(N693K).

The mutation of the present invention may be a frameshift at amino acidposition 730, 391, 461, 441, 235, 254, 564, 662, 715, 405, 685, 64, 73,656, 718, 374, 592, 505, 730, or 363 of SEQ ID NO: 3, 5 or 11 or thecorresponding nucleotide position of the nucleic acid sequence encodingSEQ ID NO: 3, 5, or 11. The mutation of the EZH2 may also be aninsertion of a glutamic acid (E) between amino acid positions 148 and149 of SEQ ID NO: 3, 5 or 21. Another example of EZH2 mutation is adeletion of glutamic acid (E) and leucine (L) at amino acid positions148 and 149 of SEQ ID NO: 3, 5 or 11. The mutant EZH2 may furthercomprise a nonsense mutation at amino acid position 733, 25, 317, 62,553, 328, 58, 207, 123, 63, 137, or 60 of SEQ ID NO: 3, 5 or 11.

Cells heterozygous for EZH2 would be expected to display a malignantphenotype due to the efficient formation of H3-K27me1 by the WT enzymeand the efficient, subsequent transition of this progenitor species toH3-K27me2, and, especially, H3-K27me3, by the mutant enzyme form(s).

Previous results point to dependency on enzymatic coupling betweenenzymes that perform H3-K27 mono-methylation and certain mutant forms ofEZH2 for pathogenesis in follicular lymphoma and diffuse large B-celllymphoma. For example, cells expressing Y641 mutant EZH2 may be moresensitive to small molecule EZH2 inhibitors than cells expressing WTEZH2. Specifically, cells expressing Y641 mutant EZH2 show reducedgrowing, dividing or proliferation, or even undergo apoptosis ornecrosis after the treatment of EZH2 inhibitors. In contrast, cellsexpressing WT EZH2 are not responsive to the anti-proliferative effectof the EZH2 inhibitors (U.S. Patent Application No. 61/381,684;incorporated herein by reference in its entirety.)

An aspect of the present invention is a method for treating oralleviating a symptom of cancer or precancerous condition in a subjectby administering to a subject expressing a mutant EZH2 comprising amutation in the substrate pocket domain as defined in SEQ ID NO: 6 atherapeutically effective amount of an EZH2 inhibitor as describedherein, e.g., a compound of Formula (IIa) in combination with anotheragent suitable to be administered together simultaneously, sequentially,or in alternation.

Another aspect of the invention is a method for inhibiting in a subjectconversion of H3-K27 to trimethylated H3-K27. The inhibition can involveinhibiting in a subject conversion of unmethylated H3-K27 tomonomethylated H3-K27, conversion of monomethylated H3-K27 todimethylated H3-K27, conversion of dimethylated H3-K27 to trimethylatedH3-K27, or any combination thereof, including, for example, conversionof monomethylated H3-K27 to dimethylated H3-K27 and conversion ofdimethylated H3-K27 to trimethylated H3-K27. As used herein,unmethylated H3-K27 refers to histone H3 with no methyl group covalentlylinked to the amino group of lysine 27. As used herein, monomethylatedH3-K27 refers to histone H3 with a single methyl group covalently linkedto the amino group of lysine 27. Monomethylated H3-K27 is also referredto herein as H3-K27me1. As used herein, dimethylated H3-K27 refers tohistone H3 with two methyl groups covalently linked to the amino groupof lysine 27. Dimethylated H3-K27 is also referred to herein asH3-K27me2. As used herein, trimethylated H3-K27 refers to histone H3with three methyl groups covalently linked to the amino group of lysine27. Trimethylated H3-K27 is also referred to herein as H3-K27me3.

Histone H3 is a 136 amino acid long protein, the sequence of which isknown. See, for example, GenBank Accession No. CAB02546, the content ofwhich is incorporated herein by reference. As disclosed further herein,in addition to full-length histone H3, peptide fragments of histone H3comprising the lysine residue corresponding to K27 of full-lengthhistone H3 can be used as substrate for EZH2 (and likewise for mutantforms of EZH2) to assess conversion of H3-K27 ml to H3-K27m2 andconversion of H3-K27m2 to H3-K27m3. In one embodiment, such peptidefragment corresponds to amino acid residues 21-44 of histone H3. Suchpeptide fragment has the amino acid sequence LATKAARKSAPATGGVKKPHRYRP(SEQ ID NO: 10).

A composition of the present invention comprises a compound of Formula(IIa) and one or more other therapeutic agents, or a pharmaceuticallyacceptable salt thereof. The compounds of Formula (IIa) are suitable foradministration as part of a combination therapy with one or more othertherapeutic agents or treatment modality, suitable to be administeredtogether, sequentially, or in alternation. Other compounds of Formula(IIa) suitable for the methods of the invention are described in U.S.Publication 20120264734, the contents of which are hereby incorporatedby reference in their entireties.

The present invention provides the compounds of Formula (IIa):

or a pharmaceutically acceptable salts or esters thereof, wherein R₇,R₈, R_(a), and R_(b) are defined herein.

The compounds of Formula (IIa) can include one or more of the followingfeatures:

For example, each of R_(a) and R_(b) independently is H or C₁-C₆ alkyloptionally substituted with one or more -Q₃-T₃.

For example, one of R_(a) and R_(b) is H.

For example, R_(a) and R_(b), together with the N atom to which they areattached, form a 4 to 7-membered heterocycloalkyl ring having 0 or 1additional heteroatoms to the N atom (e.g., azetidinyl, pyrrolidinyl,imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,triazolidinyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andthe like) and the ring is optionally substituted with one or more-Q₃-T₃.

For example, R_(a) and R_(b), together with the N atom to which they areattached, form azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahyrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl, or morpholinyl,and the ring is optionally substituted with one or more -Q₃-T₃.

For example, one or more -Q₃-T₃ are oxo.

For example, Q₃ is a bond or unsubstituted or substituted C₁-C₃ alkyllinker.

For example, T₃ is H, halo, 4 to 7-membered heterocycloalkyl, C₁-C₃alkyl, OR_(d), COOR_(d), —S(O)₂R_(d), or —NR_(d)R_(e).

For example, each of R_(d) and R_(e) independently being H or C₁-C₆alkyl.

For example, R₇ is C₃-C₈ cycloalkyl or 4 to 7-membered heterocycloalkyl,each optionally substituted with one or more -Q₅-T₅.

For example, R₇ is piperidinyl, tetrahydropyran,tetrahydro-2H-thiopyranyl, cyclopentyl, cyclohexyl, pyrrolidinyl, orcycloheptyl, each optionally substituted with one or more -Q₅-T₅.

For example, R₇ is cyclopentyl cyclohexyl or tetrahydro-2H-thiopyranyl,each of which is optionally substituted with one or more -Q₅-T₅.

For example, Q₅ is NHC(O) and T₅ is C₁-C₆ alkyl or C₁-C₆ alkoxy, each

For example, one or more -Q₅-T₅ are oxo.

For example, R₇ is 1-oxide-tetrahydro-2H-thiopyranyl or1,1-dioxide-tetrahydro-2H-thiopyranyl.

For example, Q₅ is a bond and T₅ is amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino.

For example, Q₅ is CO, S(O)₂, or NHC(O); and T₅ is C₁-C₆ alkyl, C₁-C₆alkoxyl, C₃-C₈ cycloalkyl, or 4 to 7-membered heterocycloalkyl.

For example, R₈ is H or C₁-C₆ alkyl which is optionally substituted withone or more substituents selected from the group consisting of halo,hydroxyl, COOH, C(O)O—C₁-C₆ alkyl, cyano, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, and di-C₁-C₆ alkylamino.

For example, R₈ is H, methyl, or ethyl.

In one embodiment, the compound of the invention is Compound 44

or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound of the invention is the compound itself,i.e., the free base or “naked” molecule. In another embodiment, thecompound is a salt thereof, e.g., a mono-HCl or tri-HCl salt, mono-HBror tri-HBr salt of the naked molecule.

Representative compounds of the present invention include compoundslisted in Table 1.

In the table below, each occurrence of

should be construed as

TABLE 1 Compound Number Structure MS (M + 1)⁺ 1

501.39 2

543.22 3

486.21 4

529.30 11

558.45 12

559.35 13

517.3  14

557.4  16

515.4  20

614.4  21

614.4  27

516.35 36

557.35 39

572.35 40

572.35 42

572.4  43

572.6  44

573.40 47

530.35 59

587.40 60

601.30 61

599.35 62

601.35 63

613.35 65

531.30 66

586.40 67

585.25 68

585.35 69

557.25 70

573.40 71

573.40 72

575.35 73

572.10 74

575.35 75

571.25 76

587.40 77

587.45 78

587.20 79

589.35 80

589.30 81

607.35 82

543.40 83

559.80 84

561.25 85

86

585.37 87

600.30 88

587.40 89

503.40 90

517.30 91

531.35 92

545.40 93

557.35 94

559.20 95

599.35 (M + Na) 96

577.25 97

571.40 98

547.35 99

561.30 100

591.25 101

546.35 102

560.20 103

567.30 104

585.25 105

585.40 107

108

530.35 114

573.25 115

642.45 116

545.15 117

489.20 119

609.35 122

587.55 124

650.85 125

614.75 126

572.35 127

656.65 128

586.45 129

628.35 130

591.2  131

587.35 132

589.25 133

605.25 135

621.40 136

621.45 137

589.35 138

627.5  141

614.65 142

603.45 143

578.35 144

609.15 146

641.50 178

593.60

As used herein, “alkyl”, “C₁, C₂, C₃, C₄, C₅ or C₆ alkyl” or “C₁-C₆alkyl” is intended to include C₁, C₂, C₃, C₄, C₅ or C₆ straight chain(linear) saturated aliphatic hydrocarbon groups and C₃, C₄, C₅ or C₆branched saturated aliphatic hydrocarbon groups. For example, C₁-C₆alkyl is intended to include C₁, C₂, C₃, C₄, C₅ and C₆ alkyl groups.Examples of alkyl include, moieties having from one to six carbon atoms,such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, s-pentyl or n-hexyl.

In certain embodiments, a straight chain or branched alkyl has six orfewer carbon atoms (e.g., C₁-C₆ for straight chain, C₃-C₆ for branchedchain), and in another embodiment, a straight chain or branched alkylhas four or fewer carbon atoms.

As used herein, the term “cycloalkyl” refers to a saturated orunsaturated nonaromatic hydrocarbon mono- or multi-ring (e.g., fused,bridged, or spiro rings) system having 3 to 30 carbon atoms (e.g.,C₃-C₁₀). Examples of cycloalkyl include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and adamantyl.The term “heterocycloalkyl” refers to a saturated or unsaturatednonaromatic 3-8 membered monocyclic, 7-12 membered bicyclic (fused,bridged, or spiro rings), or 11-14 membered tricyclic ring system(fused, bridged, or spiro rings) having one or more heteroatoms (such asO, N, S, or Se), unless specified otherwise. Examples ofheterocycloalkyl groups include, but are not limited to, piperidinyl,piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl,indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,triazolidinyl, tetrahyrofuranyl, oxiranyl, azetidinyl, oxetanyl,thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl,dihydropyranyl, pyranyl, morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl,2-oxa-6-azaspiro[3.3]heptanyl, 2,6-diazaspiro[3.3]heptanyl,1,4-dioxa-8-azaspiro[4.5]decanyl and the like.

The term “optionally substituted alkyl” refers to unsubstituted alkyl oralkyl having designated substituents replacing one or more hydrogenatoms on one or more carbons of the hydrocarbon backbone. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

An “arylalkyl” or an “aralkyl” moiety is an alkyl substituted with anaryl (e.g., phenylmethyl (benzyl)). An “alkylaryl” moiety is an arylsubstituted with an alkyl (e.g., methylphenyl).

As used herein, “alkyl linker” is intended to include C₁, C₂, C₃, C₄, C₅or C₆ straight chain (linear) saturated divalent aliphatic hydrocarbongroups and C₃, C₄, C₅ or C₆ branched saturated aliphatic hydrocarbongroups. For example, C₁-C₆ alkyl linker is intended to include C₁, C₂,C₃, C₄, C₅ and C₆ alkyl linker groups. Examples of alkyl linker include,moieties having from one to six carbon atoms, such as, but not limitedto, methyl (—CH₂—), ethyl (—CH₂CH₂—), n-propyl (—CH₂CH₂CH₂—), i-propyl(—CHCH₃CH₂—), n-butyl (—CH₂CH₂CH₂CH₂—), s-butyl (—CHCH₃CH₂CH₂—), i-butyl(—C(CH₃)₂CH₂—), n-pentyl (—CH₂CH₂CH₂CH₂CH₂—), s-pentyl(—CHCH₃CH₂CH₂CH₂—) or n-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₂—).

“Alkenyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double bond. For example, the term “alkenyl” includes straightchain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl,hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenylgroups. In certain embodiments, a straight chain or branched alkenylgroup has six or fewer carbon atoms in its backbone (e.g., C₂-C₆ forstraight chain, C₃-C₆ for branched chain). The term “C₂-C₆” includesalkenyl groups containing two to six carbon atoms. The term “C₃-C₆”includes alkenyl groups containing three to six carbon atoms.

The term “optionally substituted alkenyl” refers to unsubstitutedalkenyl or alkenyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

“Alkynyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but which containat least one triple bond. For example, “alkynyl” includes straight chainalkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl,heptynyl, octynyl, nonynyl, decynyl), and branched alkynyl groups. Incertain embodiments, a straight chain or branched alkynyl group has sixor fewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). The term “C₂-C₆” includes alkynyl groupscontaining two to six carbon atoms. The term “C₃-C₆” includes alkynylgroups containing three to six carbon atoms.

The term “optionally substituted alkynyl” refers to unsubstitutedalkynyl or alkynyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Other optionally substituted moieties (such as optionally substitutedcycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both theunsubstituted moieties and the moieties having one or more of thedesignated substituents. For example, substituted heterocycloalkylincludes those substituted with one or more alkyl groups, such as2,2,6,6-tetramethyl-piperidinyl and2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.

“Aryl” includes groups with aromaticity, including “conjugated,” ormulticyclic systems with at least one aromatic ring and do not containany heteroatom in the ring structure. Examples include phenyl, benzyl,1,2,3,4-tetrahydronaphthalenyl, etc.

“Heteroaryl” groups are aryl groups, as defined above, except havingfrom one to four heteroatoms in the ring structure, and may also bereferred to as “aryl heterocycles” or “heteroaromatics.” As used herein,the term “heteroaryl” is intended to include a stable 5-, 6-, or7-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclicaromatic heterocyclic ring which consists of carbon atoms and one ormore heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6heteroatoms, or e.g. 1, 2, 3, 4, 5, or 6 heteroatoms, independentlyselected from the group consisting of nitrogen, oxygen and sulfur. Thenitrogen atom may be substituted or unsubstituted (i.e., N or NR whereinR is H or other substituents, as defined). The nitrogen and sulfurheteroatoms may optionally be oxidized (i.e., N→O and S(O)_(p), wherep=1 or 2). It is to be noted that total number of S and O atoms in thearomatic heterocycle is not more than 1.

Examples of heteroaryl groups include pyrrole, furan, thiophene,thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole,oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and thelike.

Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryland heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene,benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline,naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine,indolizine.

In the case of multicyclic aromatic rings, only one of the rings needsto be aromatic (e.g., 2,3-dihydroindole), although all of the rings maybe aromatic (e.g., quinoline). The second ring can also be fused orbridged.

The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring can besubstituted at one or more ring positions (e.g., the ring-forming carbonor heteroatom such as N) with such substituents as described above, forexample, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety. Aryl and heteroarylgroups can also be fused or bridged with alicyclic or heterocyclicrings, which are not aromatic so as to form a multicyclic system (e.g.,tetralin, methylenedioxyphenyl).

As used herein, “carbocycle” or “carbocyclic ring” is intended toinclude any stable monocyclic, bicyclic or tricyclic ring having thespecified number of carbons, any of which may be saturated, unsaturated,or aromatic. Carbocycle includes cycloalkyl and aryl. For example, aC₃-C₁₄ carbocycle is intended to include a monocyclic, bicyclic ortricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbonatoms. Examples of carbocycles include, but are not limited to,cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl,cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl, adamantyl,cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl,indanyl, adamantyl and tetrahydronaphthyl. Bridged rings are alsoincluded in the definition of carbocycle, including, for example,[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane and[2.2.2]bicyclooctane. A bridged ring occurs when one or more carbonatoms link two non-adjacent carbon atoms. In one embodiment, bridgerings are one or two carbon atoms. It is noted that a bridge alwaysconverts a monocyclic ring into a tricyclic ring. When a ring isbridged, the substituents recited for the ring may also be present onthe bridge. Fused (e.g., naphthyl, tetrahydronaphthyl) and spiro ringsare also included.

As used herein, “heterocycle” or “heterocyclic group” includes any ringstructure (saturated, unsaturated, or aromatic) which contains at leastone ring heteroatom (e.g., N, O or S). Heterocycle includesheterocycloalkyl and heteroaryl. Examples of heterocycles include, butare not limited to, morpholine, pyrrolidine, tetrahydrothiophene,piperidine, piperazine, oxetane, pyran, tetrahydropyran, azetidine, andtetrahydrofuran.

Examples of heterocyclic groups include, but are not limited to,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl,pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl,pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl.

The term “substituted,” as used herein, means that any one or morehydrogen atoms on the designated atom is replaced with a selection fromthe indicated groups, provided that the designated atom's normal valencyis not exceeded, and that the substitution results in a stable compound.When a substituent is oxo or keto (i.e., =0), then 2 hydrogen atoms onthe atom are replaced. Keto substituents are not present on aromaticmoieties. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N or N═N). “Stablecompound” and “stable structure” are meant to indicate a compound thatis sufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchformula. Combinations of substituents and/or variables are permissible,but only if such combinations result in stable compounds.

When any variable (e.g., R₁) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with 0-2 R₁ moieties,then the group may optionally be substituted with up to two R₁ moietiesand R₁ at each occurrence is selected independently from the definitionof R₁. Also, combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or -0.

As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo andiodo. The term “perhalogenated” generally refers to a moiety wherein allhydrogen atoms are replaced by halogen atoms. The term “haloalkyl” or“haloalkoxyl” refers to an alkyl or alkoxyl substituted with one or morehalogen atoms.

The term “carbonyl” includes compounds and moieties which contain acarbon connected with a double bond to an oxygen atom. Examples ofmoieties containing a carbonyl include, but are not limited to,aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “carboxyl” refers to —COOH or its C₁-C₆ alkyl ester.

“Acyl” includes moieties that contain the acyl radical (R—C(O)—) or acarbonyl group. “Substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by, for example, alkyl groups,alkynyl groups, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

“Aroyl” includes moieties with an aryl or heteroaromatic moiety bound toa carbonyl group. Examples of aroyl groups include phenylcarboxy,naphthyl carboxy, etc.

“Alkoxyalkyl,” “alkylaminoalkyl,” and “thioalkoxyalkyl” include alkylgroups, as described above, wherein oxygen, nitrogen, or sulfur atomsreplace one or more hydrocarbon backbone carbon atoms.

The term “alkoxy” or “alkoxyl” includes substituted and unsubstitutedalkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom.Examples of alkoxy groups or alkoxyl radicals include, but are notlimited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxygroups. Examples of substituted alkoxy groups include halogenated alkoxygroups. The alkoxy groups can be substituted with groups such asalkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

The term “ether” or “alkoxy” includes compounds or moieties whichcontain an oxygen bonded to two carbon atoms or heteroatoms. Forexample, the term includes “alkoxyalkyl,” which refers to an alkyl,alkenyl, or alkynyl group covalently bonded to an oxygen atom which iscovalently bonded to an alkyl group.

The term “ester” includes compounds or moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc.

The term “thioalkyl” includes compounds or moieties which contain analkyl group connected with a sulfur atom. The thioalkyl groups can besubstituted with groups such as alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, carboxyacid, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “thioether” includes moieties which contain a sulfur atombonded to two carbon atoms or heteroatoms. Examples of thioethersinclude, but are not limited to alkthioalkyls, alkthioalkenyls, andalkthioalkynyls. The term “alkthioalkyls” include moieties with analkyl, alkenyl, or alkynyl group bonded to a sulfur atom which is bondedto an alkyl group. Similarly, the term “alkthioalkenyls” refers tomoieties wherein an alkyl, alkenyl or alkynyl group is bonded to asulfur atom which is covalently bonded to an alkenyl group; andalkthioalkynyls” refers to moieties wherein an alkyl, alkenyl or alkynylgroup is bonded to a sulfur atom which is covalently bonded to analkynyl group.

As used herein, “amine” or “amino” refers to unsubstituted orsubstituted —NH₂. “Alkylamino” includes groups of compounds whereinnitrogen of —NH₂ is bound to at least one alkyl group. Examples ofalkylamino groups include benzylamino, methylamino, ethylamino,phenethylamino, etc. “Dialkylamino” includes groups wherein the nitrogenof —NH₂ is bound to at least two additional alkyl groups. Examples ofdialkylamino groups include, but are not limited to, dimethylamino anddiethylamino. “Arylamino” and “diarylamino” include groups wherein thenitrogen is bound to at least one or two aryl groups, respectively.“Aminoaryl” and “aminoaryloxy” refer to aryl and aryloxy substitutedwith amino. “Alkylarylamino,” “alkylaminoaryl” or “arylaminoalkyl”refers to an amino group which is bound to at least one alkyl group andat least one aryl group. “Alkaminoalkyl” refers to an alkyl, alkenyl, oralkynyl group bound to a nitrogen atom which is also bound to an alkylgroup. “Acylamino” includes groups wherein nitrogen is bound to an acylgroup. Examples of acylamino include, but are not limited to,alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.

The term “amide” or “aminocarboxy” includes compounds or moieties thatcontain a nitrogen atom that is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups thatinclude alkyl, alkenyl or alkynyl groups bound to an amino group whichis bound to the carbon of a carbonyl or thiocarbonyl group. It alsoincludes “arylaminocarboxy” groups that include aryl or heteroarylmoieties bound to an amino group that is bound to the carbon of acarbonyl or thiocarbonyl group. The terms “alkylaminocarboxy”,“alkenylaminocarboxy”, “alkynylaminocarboxy” and “arylaminocarboxy”include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties,respectively, are bound to a nitrogen atom which is in turn bound to thecarbon of a carbonyl group. Amides can be substituted with substituentssuch as straight chain alkyl, branched alkyl, cycloalkyl, aryl,heteroaryl or heterocycle. Substituents on amide groups may be furthersubstituted.

Compounds of the present invention that contain nitrogens can beconverted to N-oxides by treatment with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to affordother compounds of the present invention. Thus, all shown and claimednitrogen-containing compounds are considered, when allowed by valencyand structure, to include both the compound as shown and its N-oxidederivative (which can be designated as N→O or N⁺—O⁻). Furthermore, inother instances, the nitrogens in the compounds of the present inventioncan be converted to N-hydroxy or N-alkoxy compounds. For example,N-hydroxy compounds can be prepared by oxidation of the parent amine byan oxidizing agent such as m-CPBA. All shown and claimednitrogen-containing compounds are also considered, when allowed byvalency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl,3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent invention includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like. In addition, a crystal polymorphism may bepresent for the compounds represented by the formula. It is noted thatany crystal form, crystal form mixture, or anhydride or hydrate thereofis included in the scope of the present invention. Furthermore,so-called metabolite which is produced by degradation of the presentcompound in vivo is included in the scope of the present invention.

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers.” Stereoisomers that are notmirror images of one another are termed “diastereoisomers,” andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture.”

A carbon atom bonded to four nonidentical substituents is termed a“chiral center.”

“Chiral isomer” means a compound with at least one chiral center.Compounds with more than one chiral center may exist either as anindividual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture.” When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahnet al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem.Educ. 1964, 41, 116).

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds or a cycloalkyl linker (e.g.,1,3-cylcobutyl). These configurations are differentiated in their namesby the prefixes cis and trans, or Z and E, which indicate that thegroups are on the same or opposite side of the double bond in themolecule according to the Cahn-Ingold-Prelog rules.

It is to be understood that the compounds of the present invention maybe depicted as different chiral isomers or geometric isomers. It shouldalso be understood that when compounds have chiral isomeric or geometricisomeric forms, all isomeric forms are intended to be included in thescope of the present invention, and the naming of the compounds does notexclude any isomeric forms.

Furthermore, the structures and other compounds discussed in thisinvention include all atropic isomers thereof. “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), imine-enamine andenamine-enamine. An example of keto-enol equilibria is betweenpyridin-2(1H)-ones and the corresponding pyridin-2-ols, as shown below.

It is to be understood that the compounds of the present invention maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present invention, and the naming ofthe compounds does not exclude any tautomer form.

The term “crystal polymorphs”, “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or a salt or solvate thereof)can crystallize in different crystal packing arrangements, all of whichhave the same elemental composition. Different crystal forms usuallyhave different X-ray diffraction patterns, infrared spectral, meltingpoints, density hardness, crystal shape, optical and electricalproperties, stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

The compounds of Formula (IIa) disclosed herein include the compoundsthemselves, as well as their salts, their esters, their solvates, andtheir prodrugs, if applicable. A salt, for example, can be formedbetween an anion and a positively charged group (e.g., amino) on anaryl- or heteroaryl-substituted benzene compound. Suitable anionsinclude chloride, bromide, iodide, sulfate, bisulfate, sulfamate,nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate,glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate,tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, andacetate (e.g., trifluoroacetate). The term “pharmaceutically acceptableanion” refers to an anion suitable for forming a pharmaceuticallyacceptable salt. Likewise, a salt can also be formed between a cationand a negatively charged group (e.g., carboxylate) on an aryl- orheteroaryl-substituted benzene compound. Suitable cations include sodiumion, potassium ion, magnesium ion, calcium ion, and an ammonium cationsuch as tetramethylammonium ion. The aryl- or heteroaryl-substitutedbenzene compounds also include those salts containing quaternarynitrogen atoms. In the salt form, it is understood that the ratio of thecompound to the cation or anion of the salt can be 1:1, or any rationother than 1:1, e.g., 3:1, 2:1, 1:2, or 1:3.

Examples of prodrugs include esters and other pharmaceuticallyacceptable derivatives, which, upon administration to a subject, arecapable of providing active aryl- or heteroaryl-substituted benzenecompounds.

Additionally, the compounds of the present invention, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

As defined herein, the term “derivative” refers to compounds that have acommon core structure, and are substituted with various groups asdescribed herein. For example, all of the compounds represented byFormula (I) are aryl- or heteroaryl-substituted benzene compounds, andhave Formula (I) as a common core.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres include,but are not limited to, acyl sulfonimides, tetrazoles, sulfonates andphosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176,1996.

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium, and isotopes of carbon include C-13 and C-14.

Any compound of Formula (IIa) of the present invention, as describedherein, may be an EZH2 inhibitor.

In certain aspects of the invention an inhibitor of EZH2 “selectivelyinhibits” histone methyltransferase activity of the mutant EZH2 when itinhibits histone methyltransferase activity of the mutant EZH2 moreeffectively than it inhibits histone methyltransferase activity ofwild-type EZH2. For example, in one embodiment the selective inhibitorhas an IC50 for the mutant EZH2 that is at least 40 percent lower thanthe IC50 for wild-type EZH2. In one embodiment the selective inhibitorhas an IC50 for the mutant EZH2 that is at least 50 percent lower thanthe IC50 for wild-type EZH2. In one embodiment the selective inhibitorhas an IC50 for the mutant EZH2 that is at least 60 percent lower thanthe IC50 for wild-type EZH2. In one embodiment the selective inhibitorhas an IC50 for the mutant EZH2 that is at least 70 percent lower thanthe IC50 for wild-type EZH2. In one embodiment the selective inhibitorhas an IC50 for the mutant EZH2 that is at least 80 percent lower thanthe IC50 for wild-type EZH2. In one embodiment the selective inhibitorhas an IC50 for the mutant EZH2 that is at least 90 percent lower thanthe IC50 for wild-type EZH2.

In one embodiment, the selective inhibitor of a mutant EZH2 exertsessentially no inhibitory effect on wild-type EZH2.

In certain aspects of the invention the inhibitor inhibits conversion ofH3-K27me2 to H3-K27me3. In one embodiment the inhibitor is said toinhibit trimethylation of H3-K27. Since conversion of H3-K27me1 toH3-K27me2 precedes conversion of H3-K27me2 to H3-K27me3, an inhibitor ofconversion of H3-K27me1 to H3-K27me2 naturally also inhibits conversionof H3-K27me2 to H3-K27me3, i.e., it inhibits trimethylation of H3-K27.It is also possible to inhibit conversion of H3-K27me2 to H3-K27me3without inhibition of conversion of H3-K27me1 to H3-K27me2. Inhibitionof this type would also result in inhibition of trimethylation ofH3-K27, albeit without inhibition of dimethylation of H3-K27.

In one embodiment the inhibitor inhibits conversion of H3-K27me1 toH3-K27me2 and the conversion of H3-K27me2 to H3-K27me3. Such inhibitormay directly inhibit the conversion of H3-K27me1 to H3-K27me2 alone.Alternatively, such inhibitor may directly inhibit both the conversionof H3-K27me1 to H3-K27me2 and the conversion of H3-K27me2 to H3-K27me3.

In certain aspects of the invention, the inhibitor compound inhibitshistone methyltransferase activity. Inhibition of histonemethyltransferase activity can be detected using any suitable method.The inhibition can be measured, for example, either in terms of rate ofhistone methyltransferase activity or as product of histonemethyltransferase activity.

The inhibition is a measurable inhibition compared to a suitablecontrol. In one embodiment, inhibition is at least 10 percent inhibitioncompared to a suitable control. That is, the rate of enzymatic activityor the amount of product with the inhibitor is less than or equal to 90percent of the corresponding rate or amount made without the inhibitor.In various other embodiments, inhibition is at least 20, 25, 30, 40, 50,60, 70, 75, 80, 90, or 95 percent inhibition compared to a suitablecontrol. In one embodiment, inhibition is at least 99 percent inhibitioncompared to a suitable control. That is, the rate of enzymatic activityor the amount of product with the inhibitor is less than or equal to 1percent of the corresponding rate or amount made without the inhibitor.

A composition of the present invention comprises a compound of Formula(IIa), or a pharmaceutically acceptable salt thereof, and one or moreother therapeutic agents, or a pharmaceutically acceptable salt thereof.The present invention provides for the administration of a compound ofFormula (IIa) or a pharmaceutically acceptable salt thereof, and one ormore therapeutic agents or a pharmaceutically acceptable salt thereof,as a co-formulation or separate formulations, wherein the administrationof formulations is simultaneous, sequential, or in alternation. Incertain embodiments, the other therapeutic agents can be an agent thatis recognized in the art as being useful to treat the disease orcondition being treated by the composition of the present invention. Inother embodiment, the other therapeutic agent can be an agent that isnot recognized in the art as being useful to treat the disease orcondition being treated by the composition of the present invention. Inone aspect, the other therapeutic agents can be an agent that imparts abeneficial attribute to the composition of the present invention (e.g.,an agent that affects the viscosity of the composition). The beneficialattribute to the composition of the present invention includes, but isnot limited to, pharmacokinetic or pharmacodynamic co-action resultingfrom the combination of a compound of Formula (IIa) and one or moreother therapeutic agents. For example, the one or more other therapeuticagents can be anticancer agents or chemotherapeutic agents. For example,the one or more other therapeutic agents can be glucocorticoids. Forexample, the one or more other therapeutic agents can be selected fromprednisone, prednisolone, cyclophosphamide, vincristine, doxorubicin,mafosfamide, cisplatin, AraC, everolimus, decitabine, dexamethasone, orfunctional analogs, derivatives, produgs, and metabolites thereof. Inanother aspect, the other therapeutic agent can be Prednisone or itsactive metabolite, Prednisolone.

The therapeutic agents set forth below are for illustrative purposes andnot intended to be limiting. The present invention includes at least oneother therapeutic agent selected from the lists below. The presentinvention can include more than one other therapeutic agent, e.g., two,three, four, or five other therapeutic agents such that the compositionof the present invention can perform its intended function.

In one embodiment, the other therapeutic agent is an anticancer agent.In one embodiment, the anticancer agent is a compound that affectshistone modifications, such as an HDAC inhibitor. In certainembodiments, an anticancer agent is selected from the group consistingof chemotherapeutics (such as 2CdA, 5-FU, 6-Mercaptopurine, 6-TG,Abraxane™, Accutane®, Actinomycin-D, Adriamycin®, Alimta®, all-transretinoic acid, amethopterin, Ara-C, Azacitadine, BCNU, Blenoxane®,Camptosar®, CeeNU®, Clofarabine, Clolar™, Cytoxan®, daunorubicinhydrochloride, DaunoXome®, Dacogen®, DIC, Doxil®, Ellence®, Eloxatin®,Emcyt®, etoposide phosphate, Fludara®, FUDR®, Gemzar®, Gleevec®,hexamethylmelamine, Hycamtin®, Hydrea®, Idamycin®, Ifex®, ixabepilone,Ixempra®, L-asparaginase, Leukeran®, liposomal Ara-C, L-PAM, Lysodren,Matulane®, mithracin, Mitomycin-C, Myleran®, Navelbine®, Neutrexin®,nilotinib, Nipent®, Nitrogen Mustard, Novantrone®, Oncaspar®, Panretin®,Paraplatin®, Platinol®, prolifeprospan 20 with carmustine implant,Sandostatin®, Targretin®, Tasigna®, Taxotere®, Temodar®, TESPA,Trisenox®, Valstar®, Velban®, Vidaza™, vincristine sulfate, VM 26,Xeloda® and Zanosar®); biologics (such as Alpha Interferon, BacillusCalmette-Guerin, Bexxar®, Campath®, Ergamisol®, Erlotinib, Herceptin®,Interleukin-2, Iressa®, lenalidomide, Mylotarg®, Ontak®, Pegasys®,Revlimid®, Rituxan®, Tarceva™, Thalomid®, Tykerb®, Velcade® andZevalin™); corticosteroids, (such as dexamethasone sodium phosphate,DeltaSone® and Delta-Cortef®); hormonal therapies (such as Arimidex®,Aromasin®, Casodex®, Cytadren®, Eligard®, Eulexin®, Evista®, Faslodex®,Femara®, Halotestin®, Megace®, Nilandron®, Nolvadex®, Plenaxis™ andZoladex®); and radiopharmaceuticals (such as Iodotope®, Metastron®,Phosphocol® and Samarium SM-153).

In another embodiment, the other therapeutic agent is a chemotherapeuticagent (also referred to as an anti-neoplastic agent oranti-proliferative agent), selected from the group including analkylating agent; an antibiotic; an anti-metabolite; a detoxifyingagent; an interferon; a polyclonal or monoclonal antibody; an EGFRinhibitor; a HER2 inhibitor; a histone deacetylase inhibitor; a hormone;a mitotic inhibitor; an MTOR inhibitor; a multi-kinase inhibitor; aserine/threonine kinase inhibitor; a tyrosine kinase inhibitors; aVEGF/VEGFR inhibitor; a taxane or taxane derivative, an aromataseinhibitor, an anthracycline, a microtubule targeting drug, atopoisomerase poison drug, an inhibitor of a molecular target or enzyme(e.g., a kinase or a protein methyltransferase), a cytidine analoguedrug or any chemotherapeutic, anti-neoplastic or anti-proliferativeagent listed in www.cancer.org/docroot/cdg/cdg_0.asp.

Exemplary alkylating agents include, but are not limited to,cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan(Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU);dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel);ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran);carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide(Temodar); thiotepa (Thioplex); bendamustine (Treanda); or streptozocin(Zanosar).

Exemplary antibiotics include, but are not limited to, doxorubicin(Adriamycin); doxorubicin liposomal (Doxil); mitoxantrone (Novantrone);bleomycin (Blenoxane); daunorubicin (Cerubidine); daunorubicin liposomal(DaunoXome); dactinomycin (Cosmegen); epirubicin (Ellence); idarubicin(Idamycin); plicamycin (Mithracin); mitomycin (Mutamycin); pentostatin(Nipent); or valrubicin (Valstar).

Exemplary anti-metabolites include, but are not limited to, fluorouracil(Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine(Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine(Arranon); cladribine (Cladribine Novaplus); clofarabine (Clolar);cytarabine (Cytosar-U); decitabine (Dacogen); cytarabine liposomal(DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine(FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine(Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall);thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

Exemplary detoxifying agents include, but are not limited to, amifostine(Ethyol) or _(mesna) (Mesnex).

Exemplary interferons include, but are not limited to, interferonalfa-2b (Intron A) or interferon alfa-2a (Roferon-A).

Exemplary polyclonal or monoclonal antibodies include, but are notlimited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab(Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab(Vectibix); tositumomab/iodinel31 tositumomab (Bexxar); alemtuzumab(Campath); ibritumomab (Zevalin; In-111; Y-90 Zevalin); gemtuzumab(Mylotarg); eculizumab (Soliris) ordenosumab.

Exemplary EGFR inhibitors include, but are not limited to, gefitinib(Iressa); lapatinib (Tykerb); _(cetuximab) (Erbitux); _(erlotinib)(Tarceva); _(panitumumab) (Vectibix); PKI-166; canertinib (CI-1033);matuzumab (Emd7200) or EKB-569.

Exemplary HER2 inhibitors include, but are not limited to, trastuzumab(Herceptin); lapatinib (Tykerb) or AC-480.

Histone Deacetylase Inhibitors include, but are not limited to,vorinostat (Zolinza).

Exemplary hormones include, but are not limited to, tamoxifen (Soltamox;Nolvadex); raloxifene (Evista); megestrol (Megace); leuprolide (Lupron;Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole(Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane(Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole(Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone(Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin);toremifene (Fareston); _(degarelix) (Firmagon); _(nilutamide)(Nilandron); abarelix (Plenaxis); or testolactone (Teslac).

Exemplary mitotic inhibitors include, but are not limited to, paclitaxel(Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin;Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos;VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole;epothilone; vinorelbine (Navelbine); camptothecin (CPT); _(irinotecan)(Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).

Exemplary MTOR inhibitors include, but are not limited to, everolimus(Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus; orAP23573.

Exemplary VEGF/VEGFR inhibitors include, but are not limited to,bev_(acizumab) (Avastin); _(sorafenib) (Nexavar); _(sunitinib) (Sutent);ranibizumab; pegaptanib; or vandetinib.

Exemplary microtubule targeting drugs include, but are not limited to,paclitaxel, docetaxel, vincristine, vinblastin, nocodazole, epothilonesand navelbine.

Exemplary topoisomerase poison drugs include, but are not limited to,teniposide, etoposide, adriamycin, camptothecin, daunorubicin,dactinomycin, mitoxantrone, amsacrine, epirubicin and idarubicin.

Exemplary taxanes or taxane derivatives include, but are not limited to,paclitaxel and docetaxol.

Exemplary general chemotherapeutic, anti-neoplastic, anti-proliferativeagents include, but are not limited to, altretamine (Hexalen);isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin(Vesanoid); azacitidine (Vidaza); bortezomib (Velcade) asparaginase(Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine(Matulane); _(pegaspargase) (Oncaspar); _(denileukin diftitox) (Ontak);_(porfimer) (Photofrin); _(aldesleukin) (Proleukin); _(lenalidomide)(Revlimid); _(bexarotene) (Targretin); _(thalidomide) (Thalomid);_(temsirolimus) (Torisel); _(arsenic trioxide) (Trisenox);_(verteporfin) (Visudyne); mimosine (Leucenol); (1M tegafur-0.4 M5-chloro-2,4-dihydroxypyrimidine-1 M potassium oxonate), or lovastatin.

In another aspect, the other therapeutic agent is a chemotherapeuticagent or a cytokine such as G-CSF (granulocyte colony stimulatingfactor).

In yet another aspect, the other therapeutic agents can be standardchemotherapy combinations such as, but not restricted to, CMF(cyclophosphamide, methotrexate and 5-fluorouracil), CAF(cyclophosphamide, adriamycin and 5-fluorouracil), AC (adriamycin andcyclophosphamide), FEC (5-fluorouracil, epirubicin, andcyclophosphamide), ACT or ATC (adriamycin, cyclophosphamide, andpaclitaxel), rituximab, Xeloda (capecitabine), Cisplatin (CDDP),Carboplatin, TS-1 (tegafur, gimestat and otastat potassium at a molarratio of 1:0.4:1), Camptothecin-11 (CPT-11, Irinotecan or Camptosar™),CHOP (cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisone orprednisolone), R—CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin,oncovin, prednisone or prednisolone), or CMFP (cyclophosphamide,methotrexate, 5-fluorouracil and prednisone).

In another aspect, the other therapeutic agents can be an inhibitor ofan enzyme, such as a receptor or non-receptor kinase. Receptor andnon-receptor kinases are, for example, tyrosine kinases orserine/threonine kinases. Kinase inhibitors described herein are smallmolecules, polynucleic acids, polypeptides, or antibodies.

Exemplary kinase inhibitors include, but are not limited to, Bevacizumab(targets VEGF), BIBW 2992 (targets EGFR and Erb2), Cetuximab/Erbitux(targets Erb 1), Imatinib/Gleevic (targets Bcr-Abl), Trastuzumab(targets Erb2), Gefitinib/Iressa (targets EGFR), Ranibizumab (targetsVEGF), Pegaptanib (targets VEGF), Erlotinib/Tarceva (targets Erb 1),Nilotinib (targets Bcr-Abl), Lapatinib (targets Erb 1 and Erb2/Her2),GW-572016/lapatinib ditosylate (targets HER2/Erb2), Panitumumab/Vectibix(targets EGFR), Vandetinib (targets RET/VEGFR), E7080 (multiple targetsincluding RET and VEGFR), Herceptin (targets HER2/Erb2), PKI-166(targets EGFR), Canertinib/CI-1033 (targets EGFR),Sunitinib/SU-11464/Sutent (targets EGFR and FLT3), Matuzumab/Emd7200(targets EGFR), EKB-569 (targets EGFR), Zd6474 (targets EGFR and VEGFR),PKC-412 (targets VEGR and FLT3), Vatalanib/Ptk787/ZK222584 (targetsVEGR), CEP-701 (targets FLT3), SU5614 (targets FLT3), MLN518 (targetsFLT3), XL999 (targets FLT3), VX-322 (targets FLT3), Azd0530 (targetsSRC), BMS-354825 (targets SRC), SKI-606 (targets SRC), CP-690 (targetsJAK), AG-490 (targets JAK), WHI-P 154 (targets JAK), WHI-P 131 (targetsJAK), sorafenib/Nexavar (targets RAF kinase, VEGFR-1, VEGFR-2, VEGFR-3,PDGFR-β, KIT, FLT-3, and RET), Dasatinib/Sprycel (BCR/ABL and Src),AC-220 (targets Flt3), AC-480 (targets all HER proteins, “panHER”),Motesanib diphosphate (targets VEGF 1-3, PDGFR, and c-kit), Denosumab(targets RANKL, inhibits SRC), AMG888 (targets HER3), and AP24534(multiple targets including Flt3).

Exemplary serine/threonine kinase inhibitors include, but are notlimited to, Rapamune (targets mTOR/FRAP1), Deforolimus (targets mTOR),Certican/Everolimus (targets mTOR/FRAP1), AP23573 (targets mTOR/FRAP1),Eril/Fasudil hydrochloride (targets RHO), Flavopiridol (targets CDK),Seliciclib/CYC202/Roscovitrine (targets CDK), SNS-032/BMS-387032(targets CDK), Ruboxistaurin (targets PKC), Pkc412 (targets PKC),Bryostatin (targets PKC), KAI-9803 (targets PKC), SF 1126 (targetsP13K), VX-680 (targets Aurora kinase), Azdl 152 (targets Aurora kinase),Arry-142886/AZD-6244 (targets MAP/MEK), SCIO-469 (targets MAP/MEK),GW681323 (targets MAP/MEK), CC-401 (targets JNK), CEP-1347 (targetsJNK), and PD 332991 (targets CDK).

Exemplary tyrosine kinase inhibitors include, but are not limited to,erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib(Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab(Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux);panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath);_(gemtuzumab) (Mylotarg); _(temsirolimus) (Torisel); _(pazopanib)(Votrient); _(dasatinib) (Sprycel); nilotinib (Tasigna); vatalanib(Ptk787; ZK222584); CEP-701; SU5614; MLN518; XL999; VX-322; Azd0530;BMS-354825; SKI-606 CP-690; AG-490; WHI-P154; WHI-P131; AC-220; orAMG888.

The present invention provides methods for combination therapy in whicha composition comprising a compound of Formula (IIa) or apharmaceutically acceptable salt thereof, and one or more othertherapeutic agents are administered to a subject in need for treatmentof a disease or cancer. The combination therapy can also be administeredto cancer cells to inhibit proliferation or induce cell death. In oneaspect, a compound of Formula (IIa) or a pharmaceutically acceptablesalt thereof is administered prior to administration of the compositionof the present invention comprising a compound of Formula (IIa) or apharmaceutically acceptable salt thereof, and one or more othertherapeutic agents. In one aspect, a compound of Formula (IIa) or apharmaceutically acceptable salt thereof is administered prior toadministration of one or more therapeutic agents, such that the othertherapeutic agents are administered either in a single composition or intwo or more compositions, e.g. administered simultaneously,sequentially, or in alternation.

In one embodiment, a composition of the present invention includes acompound of Formula (IIa) or a pharmaceutically acceptable salt thereof,and one or more anticancer agents, e.g., CHOP (cyclophosphamide,hydroxydaunorubicin, oncovin, and prednisone or prednisolone) or R-CHOP(rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin, prednisoneor prednisolone). In one embodiment, a composition of the presentinvention includes a compound of Formula (IIa) or a pharmaceuticallyacceptable salt thereof, and prednisone or prednisolone. Methods of thepresent invention include the combination therapy of administering acompound of Formula (IIa) or a pharmaceutically acceptable salt thereof,and anticancer agents, wherein the anticancer agents are CHOP, R-CHOP,prednisone, or prednisolone.

In certain embodiments, “combination therapy” is intended to embraceadministration of these therapeutic agents in a sequential manner,wherein each therapeutic agent is administered at a different time, aswell as administration of these therapeutic agents, or at least two ofthe therapeutic agents concurrently, or in a substantially simultaneousmanner. Simultaneous administration can be accomplished, for example, byadministering to the subject a single capsule having a fixed ratio ofeach therapeutic agent or in multiple, single capsules for each of thetherapeutic agents. Sequential or substantially simultaneousadministration of each therapeutic agent can be effected by anyappropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. The therapeutic agents can be administered bythe same route or by different routes. For example, a first therapeuticagent of the combination selected may be administered by intravenousinjection while the other therapeutic agents of the combination may beadministered orally. Alternatively, for example, all therapeutic agentsmay be administered orally or all therapeutic agents may be administeredby intravenous injection. Therapeutic agents may also be administered inalternation.

In certain aspects of the invention, the combination therapies featuredin the present invention can result in a synergistic effect in thetreatment of a disease or cancer. A “synergistic effect” is defined aswhere the efficacy of a combination of therapeutic agents is greaterthan the sum of the effects of any of the agents given alone. Asynergistic effect may also be an effect that cannot be achieved byadministration of any of the compounds or other therapeutic agents assingle agents. The synergistic effect may include, but is not limitedto, an effect of treating cancer by reducing tumor size, inhibitingtumor growth, or increasing survival of the subject. The synergisticeffect may also include reducing cancer cell viability, inducing cancercell death, and inhibiting or delaying cancer cell growth.

In certain aspects of the invention “combination therapy” also embracesthe administration of the therapeutic agents as described above infurther combination with other biologically active ingredients andnon-drug therapies (e.g., surgery or radiation treatment). Where thecombination therapy further comprises a non-drug treatment, the non-drugtreatment may be conducted at any suitable time so long as a beneficialeffect from the co-action of the combination of the therapeutic agentsand non-drug treatment is achieved. For example, in appropriate cases,the beneficial effect is still achieved when the non-drug treatment istemporally removed from the administration of the therapeutic agents,perhaps by days or even weeks.

In another aspect, a composition of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof, may be administered in combination with radiationtherapy. Radiation therapy can also be administered in combination witha composition of the present invention and another chemotherapeuticagent described herein as part of a multiple agent therapy.

The present invention also provides pharmaceutical compositionscomprising a compound of Formula (IIa) or pharmaceutically acceptablesalts thereof, and one or more other therapeutic agent disclosed herein,mixed with pharmaceutically suitable carriers or excipient(s) at dosesto treat or prevent a disease or condition as described herein. In oneaspect, the present invention also provides pharmaceutical compositionscomprising any compound of Table I or pharmaceutically acceptable saltsthereof, and one or more therapeutic agents, mixed with pharmaceuticallysuitable carriers or excipient (s) at doses to treat or prevent adisease or condition as described herein. In another aspect, the presentinvention also provides pharmaceutical compositions comprising Compound44

or pharmaceutically acceptable salts thereof, and one or moretherapeutic agents, mixed with pharmaceutically suitable carriers orexcipient(s) at doses to treat or prevent a disease or condition asdescribed herein. The pharmaceutical compositions of the presentinvention can also be administered in combination with other therapeuticagents or therapeutic modalities simultaneously, sequentially, or inalternation.

Mixtures of compositions of the present invention can also beadministered to the patient as a simple mixture or in suitableformulated pharmaceutical compositions. For example, one aspect of theinvention relates to a pharmaceutical composition comprising atherapeutically effective dose of an EZH2 inhibitor of Formula (IIa), ora pharmaceutically acceptable salt, hydrate, enantiomer or stereoisomerthereof; one or more other therapeutic agent, and a pharmaceuticallyacceptable diluent or carrier.

A “pharmaceutical composition” is a formulation containing the compoundsof the present invention in a form suitable for administration to asubject. In one embodiment, the pharmaceutical composition is in bulk orin unit dosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, anions, cations, materials, compositions, carriers, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

A composition of the invention can be administered to a subject in manyof the well-known methods currently used for chemotherapeutic treatment.For example, for treatment of cancers, a compound of the invention maybe injected directly into tumors, injected into the blood stream or bodycavities or taken orally or applied through the skin with patches. Thedose chosen should be sufficient to constitute effective treatment butnot so high as to cause unacceptable side effects. The state of thedisease condition (e.g., cancer, precancer, and the like) and the healthof the patient should preferably be closely monitored during and for areasonable period after treatment.

The term “therapeutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Therapeutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician. In a preferred aspect,the disease or condition to be treated is cancer. In another aspect, thedisease or condition to be treated is a cell proliferative disorder.

In certain embodiments the therapeutically effective amount of eachpharmaceutical agent used in combination will be lower when used incombination in comparison to monotherapy with each agent alone. Suchlower therapeutically effective amount could afford for lower toxicityof the therapeutic regimen.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

The pharmaceutical compositions containing active compounds of thepresent invention may be manufactured in a manner that is generallyknown, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol and sorbitol, and sodium chloridein the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The active compounds can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

In therapeutic applications, the dosages of the EZH2 inhibitor compoundsdescribed herein, other therapeutic agents described herein,compositions comprising a compound of Formula (IIa) and one or moreother therapeutic agents, or the pharmaceutical compositions used inaccordance with the invention vary depending on the agent, the age,weight, and clinical condition of the recipient patient, and theexperience and judgment of the clinician or practitioner administeringthe therapy, among other factors affecting the selected dosage.Generally, the dose should be sufficient to result in slowing, andpreferably regressing, the growth of the tumors and also preferablycausing complete regression of the cancer. Dosages can range from about0.01 mg/kg per day to about 5000 mg/kg per day. In preferred aspects,dosages can range from about 1 mg/kg per day to about 1000 mg/kg perday. In an aspect, the dose will be in the range of about 0.1 mg/day toabout 50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day toabout 10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about1 g/day, in single, divided, or continuous doses (which dose may beadjusted for the patient's weight in kg, body surface area in m², andage in years). An effective amount of a pharmaceutical agent is thatwhich provides an objectively identifiable improvement as noted by theclinician or other qualified observer. For example, regression of atumor in a patient may be measured with reference to the diameter of atumor. Decrease in the diameter of a tumor indicates regression.Regression is also indicated by failure of tumors to reoccur aftertreatment has stopped. As used herein, the term “dosage effectivemanner” refers to amount of an active compound to produce the desiredbiological effect in a subject or cell.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The composition of the present invention is capable of further formingsalts. The composition of the present invention is capable of formingmore than one salt per molecule, e.g., mono-, di-, tri-. All of theseforms are also contemplated within the scope of the claimed invention.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the compounds of the present invention wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include, but are not limitedto, those derived from inorganic and organic acids selected from2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic,glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic,hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic,isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic,mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic,pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic,salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic,sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurringamine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

Other examples of pharmaceutically acceptable salts include hexanoicacid, cyclopentane propionic acid, pyruvic acid, malonic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, muconic acid, and the like. The present invention also encompassessalts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The composition of the present invention may also be prepared as esters,for example, pharmaceutically acceptable esters. For example, acarboxylic acid function group in a compound can be converted to itscorresponding ester, e.g., a methyl, ethyl or other ester. Also, analcohol group in a compound can be converted to its corresponding ester,e.g., acetate, propionate or other ester.

The composition of the present invention can also be prepared asprodrugs, for example, pharmaceutically acceptable prodrugs. The terms“pro-drug” and “prodrug” are used interchangeably herein and refer toany compound which releases an active parent drug in vivo. Sinceprodrugs are known to enhance numerous desirable qualities ofpharmaceuticals (e.g., solubility, bioavailability, manufacturing,etc.), the compounds of the present invention can be delivered inprodrug form. Thus, the present invention is intended to cover prodrugsof the presently claimed compounds, methods of delivering the same andcompositions containing the same. “Prodrugs” are intended to include anycovalently bonded carriers that release an active parent drug of thepresent invention in vivo when such prodrug is administered to asubject. Prodrugs in the present invention are prepared by modifyingfunctional groups present in the compound in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound. Prodrugs include compounds of the present inventionwherein a hydroxy, amino, sulfhydryl, carboxy or carbonyl group isbonded to any group that may be cleaved in vivo to form a free hydroxyl,free amino, free sulfhydryl, free carboxy or free carbonyl group,respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, esters (e.g., ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds of theinvention, and the like, See Bundegaard, H., Design of Prodrugs, p 1-92,Elesevier, New York-Oxford (1985).

The composition, or pharmaceutically acceptable salts, esters orprodrugs thereof, are administered orally, nasally, transdermally,pulmonary, inhalationally, buccally, sublingually, intraperintoneally,subcutaneously, intramuscularly, intravenously, rectally,intrapleurally, intrathecally and parenterally. In one embodiment, thecompound is administered orally. One skilled in the art will recognizethe advantages of certain routes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counter,or arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the invention can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Inan embodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present invention areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentinvention. The examples do not limit the claimed invention. Based on thepresent disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the present invention.

The present invention provides compositions and methods for treatingconditions and diseases the course of which can be influenced bymodulating the methylation status of histones or other proteins, whereinsaid methylation status is mediated at least in part by the activity ofEZH2. Modulation of the methylation status of histones can in turninfluence the level of expression of target genes activated bymethylation, and/or target genes suppressed by methylation. The methodincludes administering to a subject in need of such treatment, atherapeutically effective amount of a composition of the presentinvention or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, to a subject in need of such treatment.

Based at least on the fact that abnormal histone methylation has beenfound to be associated with certain cancers and precancerous conditions,a method for treating cancer or a precancerous condition with a mutantEZH2 in a subject comprises administering to the subject in need thereofa therapeutically effective amount of a compound that inhibitsmethylation. In one embodiment a method for treating cancer or aprecancerous condition in a subject comprises administering to thesubject in need thereof a therapeutically effective amount of a compoundthat inhibits conversion of unmethylated H3-K27 to monomethylated H3-K27(H3-K27me1). In one embodiment a method for treating cancer or aprecancerous condition in a subject comprises administering to thesubject in need thereof a therapeutically effective amount of a compoundthat inhibits conversion of monomethylated H3-K27 (H3-K27me1) todimethylated H3-K27 (H3-K27me2). In one embodiment a method for treatingcancer or a precancerous condition in a subject comprises administeringto the subject in need thereof a therapeutically effective amount of acompound that inhibits conversion of H3-K27me2 to trimethylated H3-K27(H3-K27me3). In one embodiment a method for treating cancer or aprecancerous condition in a subject comprises administering to thesubject in need thereof a therapeutically effective amount of a compoundthat inhibits both conversion of H3-K27me1 to H3-K27me2 and conversionof H3-K27me2 to H3-K27me3. It is important to note that disease-specificincrease in methylation can occur at chromatin in key genomic loci inthe absence of a global increase in cellular levels of histone orprotein methylation. For example, it is possible for aberranthypermethylation at key disease-relevant genes to occur against abackdrop of global histone or protein hypomethylation.

Modulators of methylation can be used for modulating cell proliferation,generally. For example, in some cases excessive proliferation may bereduced with agents that decrease methylation, whereas insufficientproliferation may be stimulated with agents that increase methylation.Accordingly, diseases that may be treated include hyperproliferativediseases, such as benign cell growth and malignant cell growth (cancer).

The disorder in which EZH2-mediated protein methylation plays a part canbe cancer, a cell proliferative disorder, or a precancerous condition.The present invention further provides the use of a composition of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, to a subject in need of suchtreatment, for the preparation of a medicament useful for the treatmentof cancer. Exemplary cancers that may be treated include lymphomas,including non-Hodgkin lymphoma, follicular lymphoma (FL) and diffuselarge B-cell lymphoma (DLBCL); melanoma; and leukemia, including CML.Exemplary precancerous condition includes myelodisplastic syndrome (MDS;formerly known as preleukemia).

In general, compounds that are methylation modulators can be used formodulating cell proliferation, generally. For example, in some casesexcessive proliferation may be reduced with agents that decreasemethylation, whereas insufficient proliferation may be stimulated withagents that increase methylation. Accordingly, diseases that may betreated by the compounds of the invention include hyperproliferativediseases, such as benign cell growth and malignant cell growth.

As used herein, a “subject in need thereof” is a subject having adisorder in which EZH2-mediated protein methylation plays a part, or asubject having an increased risk of developing such disorder relative tothe population at large. A subject in need thereof can have aprecancerous condition. Preferably, a subject in need thereof hascancer. A “subject” includes a mammal. The mammal can be e.g., anymammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat, cow,horse, goat, camel, sheep or a pig. Preferably, the mammal is a human.

The subject of the present invention includes any human subject who hasbeen diagnosed with, has symptoms of, or is at risk of developing acancer or a precancerous condition. The subject of the present inventionincludes any human subject expressing a mutant EZH2. For example, amutant EZH2 comprises one or more mutations, wherein the mutation is asubstitution, a point mutation, a nonsense mutation, a missensemutation, a deletion, or an insertion or any other EZH2 mutationdescribed herein.

A subject in need thereof may have refractory or resistant cancer.“Refractory or resistant cancer” means cancer that does not respond totreatment. The cancer may be resistant at the beginning of treatment orit may become resistant during treatment. In some embodiments, thesubject in need thereof has cancer recurrence following remission onmost recent therapy. In some embodiments, the subject in need thereofreceived and failed all known effective therapies for cancer treatment.In some embodiments, the subject in need thereof received at least oneprior therapy. In certain embodiments the prior therapy is monotherapy.In certain embodiments the prior therapy is combination therapy.

In some embodiments, a subject in need thereof may have a secondarycancer as a result of a previous therapy. “Secondary cancer” meanscancer that arises due to or as a result from previous carcinogenictherapies, such as chemotherapy.

The subject may also exhibit resistance to EZH2 histonemethyltransferase inhibitors or any other therapeutic agent.

The invention also features a method of selecting a combination therapyfor a subject having cancer. The method includes the steps of: detectingone or more EZH2 mutations described herein in a sample from thesubject; and selecting, based on the presence of the one or more EZH2mutations, a combination therapy for treating cancer. In one embodiment,the therapy includes administering to the subject a composition of theinvention. In one embodiment, the method further includes administratingto the subject a therapeutically effective amount of a composition ofthe invention. An EZH2 mutation can be detected using any suitablemethod known in the art. More methods are described in U.S. patentpublication US 20130040906, which is incorporated herein by reference intheir entireties.

The methods and uses described herein may include steps of detecting oneor more EZH2 mutations described herein in a sample from a subject inneed thereof prior to and/or after the administration of a compositionof the invention (e.g., a composition comprising a compound of Formula(IIa) or pharmaceutically acceptable salts thereof, and one or moretherapeutic agents) to the subject. The presence of the one or more EZH2mutations described herein in the tested sample indicates the subject isresponsive to the combination therapy of the invention.

The present invention provides personalized medicine, treatment and/orcancer management for a subject by genetic screening of one or more EZH2mutations described herein in the subject. For example, the presentinvention provides methods for treating or alleviating a symptom ofcancer or a precancerous condition in a subject in need thereof bydetermining responsiveness of the subject to a combination therapy andwhen the subject is responsive to the combination therapy, administeringto the subject a composition of the invention. The responsiveness isdetermined by obtaining a sample from the subject and detecting one ormore EZH2 mutations described herein, and the presence of such one ormore EZH2 mutations described herein indicates that the subject isresponsive to the composition of the invention. Once the responsivenessof a subject is determined, a therapeutically effective amount of acomposition, for example, a composition comprising a compound of Formula(IIa) or pharmaceutically acceptable salts thereof, and one or moretherapeutic agents, can be administered. The therapeutically effectiveamount of a composition can be determined by one of ordinary skill inthe art.

As used herein, the term “responsiveness” is interchangeable with terms“responsive”, “sensitive”, and “sensitivity”, and it is meant that asubject is showing therapeutic responses when administered a compositionof the invention, e.g., tumor cells or tumor tissues of the subjectundergo apoptosis and/or necrosis, and/or display reduced growing,dividing, or proliferation. This term is also meant that a subject willor has a higher probability, relative to the population at large, ofshowing therapeutic responses when administered a composition of theinvention, e.g., tumor cells or tumor tissues of the subject undergoapoptosis and/or necrosis, and/or display reduced growing, dividing, orproliferation.

By “sample” it means any biological sample derived from the subject,includes but is not limited to, cells, tissues samples, body fluids(including, but not limited to, mucus, blood, plasma, serum, urine,saliva, and semen), tumor cells, and tumor tissues. Preferably, thesample is selected from bone marrow, peripheral blood cells, blood,plasma and serum. Samples can be provided by the subject under treatmentor testing. Alternatively samples can be obtained by the physicianaccording to routine practice in the art.

As used herein, the term “cell proliferative disorder” refers toconditions in which unregulated or abnormal growth, or both, of cellscan lead to the development of an unwanted condition or disease, whichmay or may not be cancerous. Exemplary cell proliferative disorders ofthe invention encompass a variety of conditions wherein cell division isderegulated. Exemplary cell proliferative disorder include, but are notlimited to, neoplasms, benign tumors, malignant tumors, pre-cancerousconditions, in situ tumors, encapsulated tumors, metastatic tumors,liquid tumors, solid tumors, immunological tumors, hematological tumors,cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidlydividing cells. The term “rapidly dividing cell” as used herein isdefined as any cell that divides at a rate that exceeds or is greaterthan what is expected or observed among neighboring or juxtaposed cellswithin the same tissue. A cell proliferative disorder includes aprecancer or a precancerous condition. A cell proliferative disorderincludes cancer. Preferably, the methods provided herein are used totreat or alleviate a symptom of cancer. The term “cancer” includes solidtumors, as well as, hematologic tumors and/or malignancies. A “precancercell” or “precancerous cell” is a cell manifesting a cell proliferativedisorder that is a precancer or a precancerous condition. A “cancercell” or “cancerous cell” is a cell manifesting a cell proliferativedisorder that is a cancer. Any reproducible means of measurement may beused to identify cancer cells or precancerous cells. Cancer cells orprecancerous cells can be identified by histological typing or gradingof a tissue sample (e.g., a biopsy sample). Cancer cells or precancerouscells can be identified through the use of appropriate molecularmarkers.

Exemplary non-cancerous conditions or disorders include, but are notlimited to, rheumatoid arthritis; inflammation; autoimmune disease;lymphoproliferative conditions; acromegaly; rheumatoid spondylitis;osteoarthritis; gout, other arthritic conditions; sepsis; septic shock;endotoxic shock; gram-negative sepsis; toxic shock syndrome; asthma;adult respiratory distress syndrome; chronic obstructive pulmonarydisease; chronic pulmonary inflammation; inflammatory bowel disease;Crohn's disease; psoriasis; eczema; ulcerative colitis; pancreaticfibrosis; hepatic fibrosis; acute and chronic renal disease; irritablebowel syndrome; pyresis; restenosis; cerebral malaria; stroke andischemic injury; neural trauma; Alzheimer's disease; Huntington'sdisease; Parkinson's disease; acute and chronic pain; allergic rhinitis;allergic conjunctivitis; chronic heart failure; acute coronary syndrome;cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter'ssyndrome; acute synovitis; muscle degeneration, bursitis; tendonitis;tenosynovitis; herniated, ruptures, or prolapsed intervertebral disksyndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonarysarcosis; bone resorption diseases, such as osteoporosis;graft-versus-host reaction; Multiple Sclerosis; lupus; fibromyalgia;AIDS and other viral diseases such as Herpes Zoster, Herpes Simplex I orII, influenza virus and cytomegalovirus; and diabetes mellitus.

Exemplary cancers include, but are not limited to, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,anorectal cancer, cancer of the anal canal, appendix cancer, childhoodcerebellar astrocytoma, childhood cerebral astrocytoma, basal cellcarcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bileduct cancer, intrahepatic bile duct cancer, bladder cancer, uringarybladder cancer, bone and joint cancer, osteosarcoma and malignantfibrous histiocytoma, brain cancer, brain tumor, brain stem glioma,cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodeimaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous systemcancer, nervous system lymphoma, central nervous system cancer, centralnervous system lymphoma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colon cancer, colorectal cancer, cutaneousT-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome,endometrial cancer, esophageal cancer, extracranial germ cell tumor,extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer,intraocular melanoma, retinoblastoma, gallbladder cancer, gastric(stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor (GIST), germ cell tumor, ovarian germ cell tumor,gestational trophoblastic tumor glioma, head and neck cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer,intraocular melanoma, ocular cancer, islet cell tumors (endocrinepancreas), Kaposi Sarcoma, kidney cancer, renal cancer, kidney cancer,laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia,chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cellleukemia, lip and oral cavity cancer, liver cancer, lung cancer,non-small cell lung cancer, small cell lung cancer, AIDS-relatedlymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma,Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular(eye) melanoma, merkel cell carcinoma, mesothelioma malignant,mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer ofthe tongue, multiple endocrine neoplasia syndrome, mycosis fungoides,myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases,chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma,chronic myeloproliferative disorders, nasopharyngeal cancer,neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer,ovarian cancer, ovarian epithelial cancer, ovarian low malignantpotential tumor, pancreatic cancer, islet cell pancreatic cancer,paranasal sinus and nasal cavity cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor, plasmacell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostatecancer, rectal cancer, renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewingfamily of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterinecancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer(melanoma), merkel cell skin carcinoma, small intestine cancer, softtissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer,supratentorial primitive neuroectodermal tumors, testicular cancer,throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer,transitional cell cancer of the renal pelvis and ureter and otherurinary organs, gestational trophoblastic tumor, urethral cancer,endometrial uterine cancer, uterine sarcoma, uterine corpus cancer,vaginal cancer, vulvar cancer, and Wilm's Tumor.

A “cell proliferative disorder of the hematologic system” is a cellproliferative disorder involving cells of the hematologic system. A cellproliferative disorder of the hematologic system can include lymphoma,leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplasia, benignmonoclonal gammopathy, lymphomatoid granulomatosis, lymphomatoidpapulosis, polycythemia vera, chronic myelocytic leukemia, agnogenicmyeloid metaplasia, and essential thrombocythemia. A cell proliferativedisorder of the hematologic system can include hyperplasia, dysplasia,and metaplasia of cells of the hematologic system. Preferably,compositions of the present invention may be used to treat a cancerselected from the group consisting of a hematologic cancer of thepresent invention or a hematologic cell proliferative disorder of thepresent invention. A hematologic cancer of the present invention caninclude multiple myeloma, lymphoma (including Hodgkin's lymphoma,non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas oflymphocytic and cutaneous origin), leukemia (including childhoodleukemia, hairy-cell leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, chronic lymphocytic leukemia, chronic myelocyticleukemia, chronic myelogenous leukemia, and mast cell leukemia), myeloidneoplasms and mast cell neoplasms.

A “cell proliferative disorder of the lung” is a cell proliferativedisorder involving cells of the lung. Cell proliferative disorders ofthe lung can include all forms of cell proliferative disorders affectinglung cells. Cell proliferative disorders of the lung can include lungcancer, a precancer or precancerous condition of the lung, benigngrowths or lesions of the lung, and malignant growths or lesions of thelung, and metastatic lesions in tissue and organs in the body other thanthe lung. Preferably, compositions of the present invention may be usedto treat lung cancer or cell proliferative disorders of the lung. Lungcancer can include all forms of cancer of the lung. Lung cancer caninclude malignant lung neoplasms, carcinoma in situ, typical carcinoidtumors, and atypical carcinoid tumors. Lung cancer can include smallcell lung cancer (“SCLC”), non-small cell lung cancer (“NSCLC”),squamous cell carcinoma, adenocarcinoma, small cell carcinoma, largecell carcinoma, adenosquamous cell carcinoma, and mesothelioma. Lungcancer can include “scar carcinoma,” bronchioalveolar carcinoma, giantcell carcinoma, spindle cell carcinoma, and large cell neuroendocrinecarcinoma. Lung cancer can include lung neoplasms having histologic andultrastructual heterogeneity (e.g., mixed cell types).

Cell proliferative disorders of the lung can include all forms of cellproliferative disorders affecting lung cells. Cell proliferativedisorders of the lung can include lung cancer, precancerous conditionsof the lung. Cell proliferative disorders of the lung can includehyperplasia, metaplasia, and dysplasia of the lung. Cell proliferativedisorders of the lung can include asbestos-induced hyperplasia, squamousmetaplasia, and benign reactive mesothelial metaplasia. Cellproliferative disorders of the lung can include replacement of columnarepithelium with stratified squamous epithelium, and mucosal dysplasia.Individuals exposed to inhaled injurious environmental agents such ascigarette smoke and asbestos may be at increased risk for developingcell proliferative disorders of the lung. Prior lung diseases that maypredispose individuals to development of cell proliferative disorders ofthe lung can include chronic interstitial lung disease, necrotizingpulmonary disease, scleroderma, rheumatoid disease, sarcoidosis,interstitial pneumonitis, tuberculosis, repeated pneumonias, idiopathicpulmonary fibrosis, granulomata, asbestosis, fibrosing alveolitis, andHodgkin's disease.

A “cell proliferative disorder of the colon” is a cell proliferativedisorder involving cells of the colon. Preferably, the cellproliferative disorder of the colon is colon cancer. Preferably,compositions of the present invention may be used to treat colon canceror cell proliferative disorders of the colon. Colon cancer can includeall forms of cancer of the colon. Colon cancer can include sporadic andhereditary colon cancers. Colon cancer can include malignant colonneoplasms, carcinoma in situ, typical carcinoid tumors, and atypicalcarcinoid tumors. Colon cancer can include adenocarcinoma, squamous cellcarcinoma, and adenosquamous cell carcinoma. Colon cancer can beassociated with a hereditary syndrome selected from the group consistingof hereditary nonpolyposis colorectal cancer, familial adenomatouspolyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndromeand juvenile polyposis. Colon cancer can be caused by a hereditarysyndrome selected from the group consisting of hereditary nonpolyposiscolorectal cancer, familial adenomatous polyposis, Gardner's syndrome,Peutz-Jeghers syndrome, Turcot's syndrome and juvenile polyposis.

Cell proliferative disorders of the colon can include all forms of cellproliferative disorders affecting colon cells. Cell proliferativedisorders of the colon can include colon cancer, precancerous conditionsof the colon, adenomatous polyps of the colon and metachronous lesionsof the colon. A cell proliferative disorder of the colon can includeadenoma. Cell proliferative disorders of the colon can be characterizedby hyperplasia, metaplasia, and dysplasia of the colon. Prior colondiseases that may predispose individuals to development of cellproliferative disorders of the colon can include prior colon cancer.Current disease that may predispose individuals to development of cellproliferative disorders of the colon can include Crohn's disease andulcerative colitis. A cell proliferative disorder of the colon can beassociated with a mutation in a gene selected from the group consistingof p53, ras, FAP and DCC. An individual can have an elevated risk ofdeveloping a cell proliferative disorder of the colon due to thepresence of a mutation in a gene selected from the group consisting ofp53, ras, FAP and DCC.

A “cell proliferative disorder of the pancreas” is a cell proliferativedisorder involving cells of the pancreas. Cell proliferative disordersof the pancreas can include all forms of cell proliferative disordersaffecting pancreatic cells. Cell proliferative disorders of the pancreascan include pancreas cancer, a precancer or precancerous condition ofthe pancreas, hyperplasia of the pancreas, and dysaplasia of thepancreas, benign growths or lesions of the pancreas, and malignantgrowths or lesions of the pancreas, and metastatic lesions in tissue andorgans in the body other than the pancreas. Pancreatic cancer includesall forms of cancer of the pancreas. Pancreatic cancer can includeductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cellcarcinoma, mucinous adenocarcinoma, osteoclast-like giant cellcarcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassifiedlarge cell carcinoma, small cell carcinoma, pancreatoblastoma, papillaryneoplasm, mucinous cystadenoma, papillary cystic neoplasm, and serouscystadenoma. Pancreatic cancer can also include pancreatic neoplasmshaving histologic and ultrastructual heterogeneity (e.g., mixed celltypes).

A “cell proliferative disorder of the prostate” is a cell proliferativedisorder involving cells of the prostate. Cell proliferative disordersof the prostate can include all forms of cell proliferative disordersaffecting prostate cells. Cell proliferative disorders of the prostatecan include prostate cancer, a precancer or precancerous condition ofthe prostate, benign growths or lesions of the prostate, and malignantgrowths or lesions of the prostate, and metastatic lesions in tissue andorgans in the body other than the prostate. Cell proliferative disordersof the prostate can include hyperplasia, metaplasia, and dysplasia ofthe prostate.

A “cell proliferative disorder of the skin” is a cell proliferativedisorder involving cells of the skin. Cell proliferative disorders ofthe skin can include all forms of cell proliferative disorders affectingskin cells. Cell proliferative disorders of the skin can include aprecancer or precancerous condition of the skin, benign growths orlesions of the skin, melanoma, malignant melanoma and other malignantgrowths or lesions of the skin, and metastatic lesions in tissue andorgans in the body other than the skin. Cell proliferative disorders ofthe skin can include hyperplasia, metaplasia, and dysplasia of the skin.

A “cell proliferative disorder of the ovary” is a cell proliferativedisorder involving cells of the ovary. Cell proliferative disorders ofthe ovary can include all forms of cell proliferative disordersaffecting cells of the ovary. Cell proliferative disorders of the ovarycan include a precancer or precancerous condition of the ovary, benigngrowths or lesions of the ovary, ovarian cancer, malignant growths orlesions of the ovary, and metastatic lesions in tissue and organs in thebody other than the ovary. Cell proliferative disorders of the skin caninclude hyperplasia, metaplasia, and dysplasia of cells of the ovary.

A “cell proliferative disorder of the breast” is a cell proliferativedisorder involving cells of the breast. Cell proliferative disorders ofthe breast can include all forms of cell proliferative disordersaffecting breast cells. Cell proliferative disorders of the breast caninclude breast cancer, a precancer or precancerous condition of thebreast, benign growths or lesions of the breast, and malignant growthsor lesions of the breast, and metastatic lesions in tissue and organs inthe body other than the breast. Cell proliferative disorders of thebreast can include hyperplasia, metaplasia, and dysplasia of the breast.

A cell proliferative disorder of the breast can be a precancerouscondition of the breast. Compositions of the present invention may beused to treat a precancerous condition of the breast. A precancerouscondition of the breast can include atypical hyperplasia of the breast,ductal carcinoma in situ (DCIS), intraductal carcinoma, lobularcarcinoma in situ (LCIS), lobular neoplasia, and stage 0 or grade 0growth or lesion of the breast (e.g., stage 0 or grade 0 breast cancer,or carcinoma in situ). A precancerous condition of the breast can bestaged according to the TNM classification scheme as accepted by theAmerican Joint Committee on Cancer (AJCC), where the primary tumor (T)has been assigned a stage of T0 or Tis; and where the regional lymphnodes (N) have been assigned a stage of N0; and where distant metastasis(M) has been assigned a stage of M0.

The cell proliferative disorder of the breast can be breast cancer.Preferably, compositions of the present invention may be used to treatbreast cancer. Breast cancer includes all forms of cancer of the breast.Breast cancer can include primary epithelial breast cancers. Breastcancer can include cancers in which the breast is involved by othertumors such as lymphoma, sarcoma or melanoma. Breast cancer can includecarcinoma of the breast, ductal carcinoma of the breast, lobularcarcinoma of the breast, undifferentiated carcinoma of the breast,cystosarcoma phyllodes of the breast, angiosarcoma of the breast, andprimary lymphoma of the breast. Breast cancer can include Stage I, II,IIIA, IIIB, IIIC and IV breast cancer. Ductal carcinoma of the breastcan include invasive carcinoma, invasive carcinoma in situ withpredominant intraductal component, inflammatory breast cancer, and aductal carcinoma of the breast with a histologic type selected from thegroup consisting of comedo, mucinous (colloid), medullary, medullarywith lymphcytic infiltrate, papillary, scirrhous, and tubular. Lobularcarcinoma of the breast can include invasive lobular carcinoma withpredominant in situ component, invasive lobular carcinoma, andinfiltrating lobular carcinoma. Breast cancer can include Paget'sdisease, Paget's disease with intraductal carcinoma, and Paget's diseasewith invasive ductal carcinoma. Breast cancer can include breastneoplasms having histologic and ultrastructual heterogeneity (e.g.,mixed cell types).

Preferably, compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph, or solvate thereof, maybe used to treat breast cancer. A breast cancer that is to be treatedcan include familial breast cancer. A breast cancer that is to betreated can include sporadic breast cancer. A breast cancer that is tobe treated can arise in a male subject. A breast cancer that is to betreated can arise in a female subject. A breast cancer that is to betreated can arise in a premenopausal female subject or a postmenopausalfemale subject. A breast cancer that is to be treated can arise in asubject equal to or older than 30 years old, or a subject younger than30 years old. A breast cancer that is to be treated has arisen in asubject equal to or older than 50 years old, or a subject younger than50 years old. A breast cancer that is to be treated can arise in asubject equal to or older than 70 years old, or a subject younger than70 years old.

A breast cancer that is to be treated can be typed to identify afamilial or spontaneous mutation in BRCA1, BRCA2, or p53. A breastcancer that is to be treated can be typed as having a HER2/neu geneamplification, as overexpressing HER2/neu, or as having a low,intermediate or high level of HER2/neu expression. A breast cancer thatis to be treated can be typed for a marker selected from the groupconsisting of estrogen receptor (ER), progesterone receptor (PR), humanepidermal growth factor receptor-2, Ki-67, CA15-3, CA 27-29, and c-Met.A breast cancer that is to be treated can be typed as ER-unknown,ER-rich or ER-poor. A breast cancer that is to be treated can be typedas ER-negative or ER-positive. ER-typing of a breast cancer may beperformed by any reproducible means. ER-typing of a breast cancer may beperformed as set forth in Onkologie 27: 175-179 (2004). A breast cancerthat is to be treated can be typed as PR-unknown, PR-rich, or PR-poor. Abreast cancer that is to be treated can be typed as PR-negative orPR-positive. A breast cancer that is to be treated can be typed asreceptor positive or receptor negative. A breast cancer that is to betreated can be typed as being associated with elevated blood levels ofCA 15-3, or CA 27-29, or both.

A breast cancer that is to be treated can include a localized tumor ofthe breast. A breast cancer that is to be treated can include a tumor ofthe breast that is associated with a negative sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with a positive sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with one or more positive axillary lymphnodes, where the axillary lymph nodes have been staged by any applicablemethod. A breast cancer that is to be treated can include a tumor of thebreast that has been typed as having nodal negative status (e.g.,node-negative) or nodal positive status (e.g., node-positive). A breastcancer that is to be treated can include a tumor of the breast that hasmetastasized to other locations in the body. A breast cancer that is tobe treated can be classified as having metastasized to a locationselected from the group consisting of bone, lung, liver, or brain. Abreast cancer that is to be treated can be classified according to acharacteristic selected from the group consisting of metastatic,localized, regional, local-regional, locally advanced, distant,multicentric, bilateral, ipsilateral, contralateral, newly diagnosed,recurrent, and inoperable.

A compound of the present invention, or a pharmaceutically acceptablesalt, ester, prodrug, metabolite, polymorph or solvate thereof, may beused to treat or prevent a cell proliferative disorder of the breast, orto treat or prevent breast cancer, in a subject having an increased riskof developing breast cancer relative to the population at large. Asubject with an increased risk of developing breast cancer relative tothe population at large is a female subject with a family history orpersonal history of breast cancer. A subject with an increased risk ofdeveloping breast cancer relative to the population at large is a femalesubject having a germ-line or spontaneous mutation in BRCA1 or BRCA2, orboth. A subject with an increased risk of developing breast cancerrelative to the population at large is a female subject with a familyhistory of breast cancer and a germ-line or spontaneous mutation inBRCA1 or BRCA2, or both. A subject with an increased risk of developingbreast cancer relative to the population at large is a female who isgreater than 30 years old, greater than 40 years old, greater than 50years old, greater than 60 years old, greater than 70 years old, greaterthan 80 years old, or greater than 90 years old. A subject with anincreased risk of developing breast cancer relative to the population atlarge is a subject with atypical hyperplasia of the breast, ductalcarcinoma in situ (DCIS), intraductal carcinoma, lobular carcinoma insitu (LCIS), lobular neoplasia, or a stage 0 growth or lesion of thebreast (e.g., stage 0 or grade 0 breast cancer, or carcinoma in situ).

A breast cancer that is to be treated can histologically gradedaccording to the Scarff-Bloom-Richardson system, wherein a breast tumorhas been assigned a mitosis count score of 1, 2, or 3; a nuclearpleiomorphism score of 1, 2, or 3; a tubule formation score of 1, 2, or3; and a total Scarff-Bloom-Richardson score of between 3 and 9. Abreast cancer that is to be treated can be assigned a tumor gradeaccording to the International Consensus Panel on the Treatment ofBreast Cancer selected from the group consisting of grade 1, grade 1-2,grade 2, grade 2-3, or grade 3.

A cancer that is to be treated can be staged according to the AmericanJoint Committee on Cancer (AJCC) TNM classification system, where thetumor (T) has been assigned a stage of TX, T1, T1mic, T1a, T1b, T1c, T2,T3, T4, T4a, T4b, T4c, or T4d; and where the regional lymph nodes (N)have been assigned a stage of NX, N0, N1, N2, N2a, N2b, N3, N3a, N3b, orN3c; and where distant metastasis (M) can be assigned a stage of MX, M0,or M1. A cancer that is to be treated can be staged according to anAmerican Joint Committee on Cancer (AJCC) classification as Stage I,Stage IIA, Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV. Acancer that is to be treated can be assigned a grade according to anAJCC classification as Grade GX (e.g., grade cannot be assessed), Grade1, Grade 2, Grade 3 or Grade 4. A cancer that is to be treated can bestaged according to an AJCC pathologic classification (pN) of pNX, pNO,PN0(I−), PN0(I+), PN0(mol−), PN0(mol+), PN1, PN1(mi), PN1a, PN1b, PN1c,pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

A cancer that is to be treated can include a tumor that has beendetermined to be less than or equal to about 2 centimeters in diameter.A cancer that is to be treated can include a tumor that has beendetermined to be from about 2 to about 5 centimeters in diameter. Acancer that is to be treated can include a tumor that has beendetermined to be greater than or equal to about 3 centimeters indiameter. A cancer that is to be treated can include a tumor that hasbeen determined to be greater than 5 centimeters in diameter. A cancerthat is to be treated can be classified by microscopic appearance aswell differentiated, moderately differentiated, poorly differentiated,or undifferentiated. A cancer that is to be treated can be classified bymicroscopic appearance with respect to mitosis count (e.g., amount ofcell division) or nuclear pleiomorphism (e.g., change in cells). Acancer that is to be treated can be classified by microscopic appearanceas being associated with areas of necrosis (e.g., areas of dying ordegenerating cells). A cancer that is to be treated can be classified ashaving an abnormal karyotype, having an abnormal number of chromosomes,or having one or more chromosomes that are abnormal in appearance. Acancer that is to be treated can be classified as being aneuploid,triploid, tetraploid, or as having an altered ploidy. A cancer that isto be treated can be classified as having a chromosomal translocation,or a deletion or duplication of an entire chromosome, or a region ofdeletion, duplication or amplification of a portion of a chromosome.

A cancer that is to be treated can be evaluated by DNA cytometry, flowcytometry, or image cytometry. A cancer that is to be treated can betyped as having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cellsin the synthesis stage of cell division (e.g., in S phase of celldivision). A cancer that is to be treated can be typed as having a lowS-phase fraction or a high S-phase fraction.

As used herein, a “normal cell” is a cell that cannot be classified aspart of a “cell proliferative disorder”. A normal cell lacks unregulatedor abnormal growth, or both, that can lead to the development of anunwanted condition or disease. Preferably, a normal cell possessesnormally functioning cell cycle checkpoint control mechanisms.

As used herein, “contacting a cell” refers to a condition in which acompound or other composition of matter is in direct contact with acell, or is close enough to induce a desired biological effect in acell.

As used herein, “candidate compound” refers to a compound of the presentinvention, or a pharmaceutically acceptable salt, ester, prodrug,metabolite, polymorph or solvate thereof, that has been or will betested in one or more in vitro or in vivo biological assays, in order todetermine if that compound is likely to elicit a desired biological ormedical response in a cell, tissue, system, animal or human that isbeing sought by a researcher or clinician. A candidate compound is acompound of the present invention, or a pharmaceutically acceptablesalt, ester, prodrug, metabolite, polymorph or solvate thereof. Thebiological or medical response can be the treatment of cancer. Thebiological or medical response can be treatment or prevention of a cellproliferative disorder. In vitro or in vivo biological assays caninclude, but are not limited to, enzymatic activity assays,electrophoretic mobility shift assays, reporter gene assays, in vitrocell viability assays, and the assays described herein.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, to alleviate the symptoms or complicationsof a disease, condition or disorder, or to eliminate the disease,condition or disorder.

A composition of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, can also beused to prevent a disease, condition or disorder. As used herein,“preventing” or “prevent” describes reducing or eliminating the onset ofthe symptoms or complications of the disease, condition or disorder.

As used herein, the term “alleviate” is meant to describe a process bywhich the severity of a sign or symptom of a disorder is decreased.Importantly, a sign or symptom can be alleviated without beingeliminated. In a preferred embodiment, the administration ofpharmaceutical compositions of the invention leads to the elimination ofa sign or symptom, however, elimination is not required. Effectivedosages are expected to decrease the severity of a sign or symptom. Forinstance, a sign or symptom of a disorder such as cancer, which canoccur in multiple locations, is alleviated if the severity of the canceris decreased within at least one of multiple locations.

As used herein, the term “severity” is meant to describe the potentialof cancer to transform from a precancerous, or benign, state into amalignant state. Alternatively, or in addition, severity is meant todescribe a cancer stage, for example, according to the TNM system(accepted by the International Union Against Cancer (UICC) and theAmerican Joint Committee on Cancer (AJCC)) or by other art-recognizedmethods. Cancer stage refers to the extent or severity of the cancer,based on factors such as the location of the primary tumor, tumor size,number of tumors, and lymph node involvement (spread of cancer intolymph nodes). Alternatively, or in addition, severity is meant todescribe the tumor grade by art-recognized methods (see, National CancerInstitute, www.cancer.gov). Tumor grade is a system used to classifycancer cells in terms of how abnormal they look under a microscope andhow quickly the tumor is likely to grow and spread. Many factors areconsidered when determining tumor grade, including the structure andgrowth pattern of the cells. The specific factors used to determinetumor grade vary with each type of cancer. Severity also describes ahistologic grade, also called differentiation, which refers to how muchthe tumor cells resemble normal cells of the same tissue type (see,National Cancer Institute, www.cancer.gov). Furthermore, severitydescribes a nuclear grade, which refers to the size and shape of thenucleus in tumor cells and the percentage of tumor cells that aredividing (see, National Cancer Institute, www.cancer.gov).

In another aspect of the invention, severity describes the degree towhich a tumor has secreted growth factors, degraded the extracellularmatrix, become vascularized, lost adhesion to juxtaposed tissues, ormetastasized. Moreover, severity describes the number of locations towhich a primary tumor has metastasized. Finally, severity includes thedifficulty of treating tumors of varying types and locations. Forexample, inoperable tumors, those cancers which have greater access tomultiple body systems (hematological and immunological tumors), andthose which are the most resistant to traditional treatments areconsidered most severe. In these situations, prolonging the lifeexpectancy of the subject and/or reducing pain, decreasing theproportion of cancerous cells or restricting cells to one system, andimproving cancer stage/tumor grade/histological grade/nuclear grade areconsidered alleviating a sign or symptom of the cancer.

As used herein the term “symptom” is defined as an indication ofdisease, illness, injury, or that something is not right in the body.Symptoms are felt or noticed by the individual experiencing the symptom,but may not easily be noticed by others. Others are defined asnon-health-care professionals.

As used herein the term “sign” is also defined as an indication thatsomething is not right in the body. But signs are defined as things thatcan be seen by a doctor, nurse, or other health care professional.

Cancer is a group of diseases that may cause almost any sign or symptom.The signs and symptoms will depend on where the cancer is, the size ofthe cancer, and how much it affects the nearby organs or structures. Ifa cancer spreads (metastasizes), then symptoms may appear in differentparts of the body.

The disorder in which EZH2-mediated protein methylation plays a part canbe a neurological disease. The compound of this invention can thus alsobe used for treating neurologic diseases such as epilepsy,schizophrenia, bipolar disorder or other psychological and/orpsychiatric disorders, neuropathies, skeletal muscle atrophy, andneurodegenerative diseases, e.g., a neurodegenerative disease. Exemplaryneurodegenerative diseases include: Alzheimer's, Amyotrophic LateralSclerosis (ALS), and Parkinson's disease. Another class ofneurodegenerative diseases includes diseases caused at least in part byaggregation of poly-glutamine. Diseases of this class include:Huntington's Diseases, Spinalbulbar Muscular Atrophy (SBMA or Kennedy'sDisease) Dentatorubropallidoluysian Atrophy (DRPLA), SpinocerebellarAtaxia 1 (SCA1), Spinocerebellar Ataxia 2 (SCA2), Machado-Joseph Disease(MJD; SCA3), Spinocerebellar Ataxia 6 (SCA6), Spinocerebellar Ataxia 7(SCA7), and Spinocerebellar Ataxia 12 (SCA12).

Any other disease in which epigenetic methylation, which is mediated byEZH2, plays a role may be treatable or preventable using compositionsand methods described herein.

Treating cancer can result in a reduction in size of a tumor. Areduction in size of a tumor may also be referred to as “tumorregression”. Preferably, after treatment, tumor size is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorsize is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Size of a tumor may be measured by any reproducible means ofmeasurement. The size of a tumor may be measured as a diameter of thetumor.

Treating cancer can result in a reduction in tumor volume. Preferably,after treatment, tumor volume is reduced by 5% or greater relative toits size prior to treatment; more preferably, tumor volume is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75% or greater. Tumor volume may bemeasured by any reproducible means of measurement.

Treating cancer results in a decrease in number of tumors. Preferably,after treatment, tumor number is reduced by 5% or greater relative tonumber prior to treatment; more preferably, tumor number is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. Number of tumors may bemeasured by any reproducible means of measurement. The number of tumorsmay be measured by counting tumors visible to the naked eye or at aspecified magnification. Preferably, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in a decrease in number of metastatic lesionsin other tissues or organs distant from the primary tumor site.Preferably, after treatment, the number of metastatic lesions is reducedby 5% or greater relative to number prior to treatment; more preferably,the number of metastatic lesions is reduced by 10% or greater; morepreferably, reduced by 20% or greater; more preferably, reduced by 30%or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. The number of metastatic lesions may be measured byany reproducible means of measurement. The number of metastatic lesionsmay be measured by counting metastatic lesions visible to the naked eyeor at a specified magnification. Preferably, the specified magnificationis 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population receivingcarrier alone. Preferably, the average survival time is increased bymore than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population ofuntreated subjects. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in increase in average survival time of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingcarrier alone. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to an untreatedpopulation. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to a populationreceiving monotherapy with a drug that is not a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof. Preferably, the mortality rate isdecreased by more than 2%; more preferably, by more than 5%; morepreferably, by more than 10%; and most preferably, by more than 25%. Adecrease in the mortality rate of a population of treated subjects maybe measured by any reproducible means. A decrease in the mortality rateof a population may be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing initiation of treatment with an active compound. A decrease inthe mortality rate of a population may also be measured, for example, bycalculating for a population the average number of disease-relateddeaths per unit time following completion of a first round of treatmentwith an active compound.

Treating cancer can result in a decrease in tumor growth rate.Preferably, after treatment, tumor growth rate is reduced by at least 5%relative to number prior to treatment; more preferably, tumor growthrate is reduced by at least 10%; more preferably, reduced by at least20%; more preferably, reduced by at least 30%; more preferably, reducedby at least 40%; more preferably, reduced by at least 50%; even morepreferably, reduced by at least 50%; and most preferably, reduced by atleast 75%. Tumor growth rate may be measured by any reproducible meansof measurement. Tumor growth rate can be measured according to a changein tumor diameter per unit time.

Treating cancer can result in a decrease in tumor regrowth. Preferably,after treatment, tumor regrowth is less than 5%; more preferably, tumorregrowth is less than 10%; more preferably, less than 20%; morepreferably, less than 30%; more preferably, less than 40%; morepreferably, less than 50%; even more preferably, less than 50%; and mostpreferably, less than 75%. Tumor regrowth may be measured by anyreproducible means of measurement. Tumor regrowth is measured, forexample, by measuring an increase in the diameter of a tumor after aprior tumor shrinkage that followed treatment. A decrease in tumorregrowth is indicated by failure of tumors to reoccur after treatmenthas stopped.

Treating or preventing a cell proliferative disorder can result in areduction in the rate of cellular proliferation. Preferably, aftertreatment, the rate of cellular proliferation is reduced by at least 5%;more preferably, by at least 10%; more preferably, by at least 20%; morepreferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The rate of cellular proliferation maybe measured by any reproducible means of measurement. The rate ofcellular proliferation is measured, for example, by measuring the numberof dividing cells in a tissue sample per unit time.

Treating or preventing a cell proliferative disorder can result in areduction in the proportion of proliferating cells. Preferably, aftertreatment, the proportion of proliferating cells is reduced by at least5%; more preferably, by at least 10%; more preferably, by at least 20%;more preferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The proportion of proliferating cellsmay be measured by any reproducible means of measurement. Preferably,the proportion of proliferating cells is measured, for example, byquantifying the number of dividing cells relative to the number ofnondividing cells in a tissue sample. The proportion of proliferatingcells can be equivalent to the mitotic index.

Treating or preventing a cell proliferative disorder can result in adecrease in size of an area or zone of cellular proliferation.Preferably, after treatment, size of an area or zone of cellularproliferation is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least 10%; more preferably,reduced by at least 20%; more preferably, reduced by at least 30%; morepreferably, reduced by at least 40%; more preferably, reduced by atleast 50%; even more preferably, reduced by at least 50%; and mostpreferably, reduced by at least 75%. Size of an area or zone of cellularproliferation may be measured by any reproducible means of measurement.The size of an area or zone of cellular proliferation may be measured asa diameter or width of an area or zone of cellular proliferation.

Treating or preventing a cell proliferative disorder can result in adecrease in the number or proportion of cells having an abnormalappearance or morphology. Preferably, after treatment, the number ofcells having an abnormal morphology is reduced by at least 5% relativeto its size prior to treatment; more preferably, reduced by at least10%; more preferably, reduced by at least 20%; more preferably, reducedby at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. An abnormalcellular appearance or morphology may be measured by any reproduciblemeans of measurement. An abnormal cellular morphology can be measured bymicroscopy, e.g., using an inverted tissue culture microscope. Anabnormal cellular morphology can take the form of nuclear pleiomorphism.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population. Thecompared populations can be cell populations. Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, acts selectively on a canceror precancerous cell but not on a normal cell. Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, acts selectively to modulateone molecular target (e.g., a target protein methyltransferase) but doesnot significantly modulate another molecular target (e.g., a non-targetprotein methyltransferase). The invention also provides a method forselectively inhibiting the activity of an enzyme, such as a proteinmethyltransferase. Preferably, an event occurs selectively in populationA relative to population B if it occurs greater than two times morefrequently in population A as compared to population B. An event occursselectively if it occurs greater than five times more frequently inpopulation A. An event occurs selectively if it occurs greater than tentimes more frequently in population A; more preferably, greater thanfifty times; even more preferably, greater than 100 times; and mostpreferably, greater than 1000 times more frequently in population A ascompared to population B. For example, cell death would be said to occurselectively in cancer cells if it occurred greater than twice asfrequently in cancer cells as compared to normal cells.

A composition of the present invention, e.g., a composition comprisingany compound of Formula (IIa) or pharmaceutically acceptable saltthereof, and one or more other therapeutic agents, such as prednisone,can modulate the activity of a molecular target (e.g., a target proteinmethyltransferase). Modulating refers to stimulating or inhibiting anactivity of a molecular target. Preferably, a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, modulates the activity of a moleculartarget if it stimulates or inhibits the activity of the molecular targetby at least 2-fold relative to the activity of the molecular targetunder the same conditions but lacking only the presence of saidcompound. More preferably, a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, modulates the activity of a molecular target if itstimulates or inhibits the activity of the molecular target by at least5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least100-fold relative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound. The activityof a molecular target may be measured by any reproducible means. Theactivity of a molecular target may be measured in vitro or in vivo. Forexample, the activity of a molecular target may be measured in vitro byan enzymatic activity assay or a DNA binding assay, or the activity of amolecular target may be measured in vivo by assaying for expression of areporter gene.

A composition of the present invention does not significantly modulatethe activity of a molecular target if the addition of the compound doesnot stimulate or inhibit the activity of the molecular target by greaterthan 10% relative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound.

As used herein, the term “isozyme selective” means preferentialinhibition or stimulation of a first isoform of an enzyme in comparisonto a second isoform of an enzyme (e.g., preferential inhibition orstimulation of a protein methyltransferase isozyme alpha in comparisonto a protein methyltransferase isozyme beta). Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, demonstrates a minimum of afourfold differential, preferably a tenfold differential, morepreferably a fifty fold differential, in the dosage required to achievea biological effect. Preferably, a compound of the present invention, ora pharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, demonstrates this differential across the range ofinhibition, and the differential is exemplified at the IC₅₀, i.e., a 50%inhibition, for a molecular target of interest.

Administering a composition of the present invention to a cell or asubject in need thereof can result in modulation (i.e., stimulation orinhibition) of an activity of a protein methyltransferase of interest.

Administering a compound of the present invention, e.g., a compositioncomprising any compound of Formula (IIa) or pharmaceutically acceptablesalt thereof, and one or more other therapeutic agents, such asprednisone, to a cell or a subject in need thereof results in modulation(i.e., stimulation or inhibition) of an activity of an intracellulartarget (e.g., substrate). Several intracellular targets can be modulatedwith the compounds of the present invention, including, but not limitedto, protein methyltrasferase.

Activating refers to placing a composition of matter (e.g., protein ornucleic acid) in a state suitable for carrying out a desired biologicalfunction. A composition of matter capable of being activated also has anunactivated state. An activated composition of matter may have aninhibitory or stimulatory biological function, or both.

Elevation refers to an increase in a desired biological activity of acomposition of matter (e.g., a protein or a nucleic acid). Elevation mayoccur through an increase in concentration of a composition of matter.

As used herein, “a cell cycle checkpoint pathway” refers to abiochemical pathway that is involved in modulation of a cell cyclecheckpoint. A cell cycle checkpoint pathway may have stimulatory orinhibitory effects, or both, on one or more functions comprising a cellcycle checkpoint. A cell cycle checkpoint pathway is comprised of atleast two compositions of matter, preferably proteins, both of whichcontribute to modulation of a cell cycle checkpoint. A cell cyclecheckpoint pathway may be activated through an activation of one or moremembers of the cell cycle checkpoint pathway. Preferably, a cell cyclecheckpoint pathway is a biochemical signaling pathway.

As used herein, “cell cycle checkpoint regulator” refers to acomposition of matter that can function, at least in part, in modulationof a cell cycle checkpoint. A cell cycle checkpoint regulator may havestimulatory or inhibitory effects, or both, on one or more functionscomprising a cell cycle checkpoint. A cell cycle checkpoint regulatorcan be a protein or not a protein.

Treating cancer or a cell proliferative disorder can result in celldeath, and preferably, cell death results in a decrease of at least 10%in number of cells in a population. More preferably, cell death means adecrease of at least 20%; more preferably, a decrease of at least 30%;more preferably, a decrease of at least 40%; more preferably, a decreaseof at least 50%; most preferably, a decrease of at least 75%. Number ofcells in a population may be measured by any reproducible means. Anumber of cells in a population can be measured by fluorescenceactivated cell sorting (FACS), immunofluorescence microscopy and lightmicroscopy. Methods of measuring cell death are as shown in Li et al.,Proc NatlAcad Sci US A. 100(5): 2674-8, 2003. In an aspect, cell deathoccurs by apoptosis.

Preferably, an effective amount of a composition of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, is not significantly cytotoxic to normalcells. A therapeutically effective amount of a compound is notsignificantly cytotoxic to normal cells if administration of thecompound in a therapeutically effective amount does not induce celldeath in greater than 10% of normal cells. A therapeutically effectiveamount of a compound does not significantly affect the viability ofnormal cells if administration of the compound in a therapeuticallyeffective amount does not induce cell death in greater than 10% ofnormal cells. In an aspect, cell death occurs by apoptosis.

Contacting a cell with a composition of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, can induce or activate cell death selectively in cancercells. Administering to a subject in need thereof a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, polymorph or solvate thereof, can induce or activate celldeath selectively in cancer cells. Contacting a cell with a compositionof the present invention, or a pharmaceutically acceptable salt,prodrug, metabolite, polymorph or solvate thereof, can induce cell deathselectively in one or more cells affected by a cell proliferativedisorder. Preferably, administering to a subject in need thereof acomposition of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, induces celldeath selectively in one or more cells affected by a cell proliferativedisorder.

The present invention relates to a method of treating or preventingcancer by administering a composition of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, polymorph orsolvate thereof, to a subject in need thereof, where administration ofthe composition of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, polymorph or solvate thereof,results in one or more of the following: prevention of cancer cellproliferation by accumulation of cells in one or more phases of the cellcycle (e.g. G1, G1/S, G2/M), or induction of cell senescence, orpromotion of tumor cell differentiation; promotion of cell death incancer cells via cytotoxicity, necrosis or apoptosis, without asignificant amount of cell death in normal cells, antitumor activity inanimals with a therapeutic index of at least 2. As used herein,“therapeutic index” is the maximum tolerated dose divided by theefficacious dose.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual (3^(rd) edition), Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18^(th) edition (1990).These texts can, of course, also be referred to in making or using anaspect of the invention.

Example 1

Synthesis ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide

Step 1: Synthesis of 5-bromo-2-methyl-3-nitrobenzoic acid

To stirred solution of 2-methyl-3-nitrobenzoic acid (100 g, 552 mmol) inconc. H2SO₄ (400 mL), 1,3-dibromo-5,5-dimethyl-2,4-imidazolidinedione(88 g, 308 mmol) was added in a portion wise manner at room temperatureand the reaction mixture was then stirred at room temperature for 5 h.The reaction mixture was poured onto ice cold water, the precipitatedsolid was filtered off, washed with water and dried under vacuum toafford the desired compound as a solid (140 g, 98%). The isolatedcompound was taken directly into the next step. ¹H NMR (DMSO-d₆, 400MHz) δ 8.31 (s, 1H), 8.17 (s, 1H), 2.43 (s, 3H).

Step 2: Synthesis of methyl 5-bromo-2-methyl-3-nitrobenzoate

To a stirred solution of 5-bromo-2-methyl-3-nitrobenzoic acid (285 g,1105 mmol) in DMF (2.8 L) at room temperature was added sodium carbonate(468 g, 4415 mmol) followed by addition of methyl iodide (626.6 g, 4415mmol). The resulting reaction mixture was heated at 60° C. for 8 h.After completion (monitored by TLC), the reaction mixture was filtered(to remove sodium carbonate) and washed with ethyl acetate (1 L×3). Thecombined filtrate was washed with water (3 L×5) and the aqueous phasewas back extracted with ethyl acetate (1 L×3). The combined organiclayers were dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure to afford the title compound as asolid (290 g, 97% yield). The isolated compound was taken directly intothe next step. ¹H NMR (CDCl₃, 400 MHz) δ 8.17 (s, 1H), 7.91 (s, 1H),3.96 (s, 3H), 2.59 (s, 3H).

Step 3: Synthesis of methyl 3-amino-5-bromo-2-methylbenzoate

To a stirred solution of methyl 5-bromo-2-methyl-3-nitrobenzoate (290 g,1058 mmol) in ethanol (1.5 L) was added aqueous ammonium chloride (283g, 5290 mmol dissolved in 1.5 L water). The resulting mixture wasstirred at 80° C. to which iron powder (472 g, 8451 mmol) was added in aportion wise manner. The resulting reaction mixture was heated at 80° C.for 12 h. Upon completion as determined by TLC, the reaction mixture washot filtered over Celite® and the celite bed was washed with methanol (5L) followed by washing with 30% MeOH in DCM (5 L). The combined filtratewas concentrated in-vacuo, the residue obtained was diluted with aqueoussodium bicarbonate solution (2 L) and extracted with ethyl acetate (5L×3). The combined organic layers were dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to afford thetitle compound as a solid (220 g, 85%). The compound was taken directlyinto the next step. ¹H NMR (CDCl₃, 400 MHz) δ 7.37 (s, 1H), 6.92 (s,1H), 3.94 (s, 3H), 3.80 (bs, 2H), 2.31 (s, 3H).

Step 4: Synthesis of methyl5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate

To a stirred solution of methyl 3-amino-5-bromo-2-methylbenzoate (15 g,61.5 mmol) and dihydro-2H-pyran-4(3)-one (9.2 g, 92 mmol) indichloroethane (300 mL) was added acetic acid (22 g, 369 mmol) and thereaction mixture stirred at room temperature for 15 minutes, then thereaction mixture was cooled to 0° C. and sodium triacetoxyborohydride(39 g, 184 mmol) was added. The reaction mixture was stirred overnightat room temperature. Upon completion of the reaction as determined byTLC, aqueous sodium bicarbonate solution was added to the reactionmixture until a pH of 7-8 was obtained. The organic phase was separatedand the aqueous phase was extracted with ethyl acetate. The combinedorganic layers were dried over anhydrous sodium sulfate, filtered andconcentrated under reduced pressure. The crude compound was purified bycolumn chromatography (100-200 mesh silica gel) eluting with ethylacetate: hexane to afford the desired compound as a solid (14 g, 69%).¹H NMR (DMSO-d₆, 400 MHz) δ 7.01 (s, 1H), 6.98 (s, 1H), 5.00 (d, 1H,J=7.6 Hz), 3.84-3.87 (m, 2H), 3.79 (s, 3H), 3.54-3.56 (m, 1H), 3.43 (t,2H, J=12 Hz), 2.14 (s, 3H), 1.81-1.84 (m, 2H), 1.47-1.55 (m, 2H).

Step 5: Synthesis of methyl 5-bromo-3-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoate

To a stirred solution of methyl5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (14 g,42.7 mmol) in dichloroethane (150 mL) was added acetaldehyde (3.75 g,85.2 mmol) and acetic acid (15.3 g, 256 mmol). The resulting reactionmixture was stirred at room temperature for 15 minutes. The mixture wascooled to 0° C. and sodium triacetoxyborohydride (27 g, 128 mmol) wasadded. The reaction mixture was stirred at room temperature for 3 hours.Upon completion of the reaction as determined by TLC, aqueous sodiumbicarbonate solution was added to the reaction mixture until a pH 7-8was obtained, the organic phase was separated and the aqueous phase wasextracted with ethyl acetate. The combined organic layers were driedover anhydrous sodium sulfate, filtered and concentrated under reducedpressure. The crude compound was purified by column chromatography(100-200 mesh silica gel) eluting with ethyl acetate: hexane to affordthe desired compound as a viscous liquid (14 g, 93%). ¹H NMR (DMSO-d₆,400 MHz) δ 7.62 (s, 1H), 7.52 (s, 1H), 3.80 (bs, 5H), 3.31 (t, 2H),2.97-3.05 (m, 2H), 2.87-2.96 (m, 1H), 2.38 (s, 3H), 1.52-1.61 (m, 2H),1.37-1.50 (m, 2H), 0.87 (t, 3H, J=6.8 Hz).

Step 6: Synthesis of 5-bromo-N-((4, 6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl)-3-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzamide

To a stirred solution of 5-bromo-3-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzoate (14 g, 39.4 mmol) in ethanol (100 mL) was addedaqueous NaOH (2.36 g, 59.2 mmol in 25 mL water) and the resultingmixture was stirred at 60° C. for 1 h. Upon completion of the reactionas determined by TLC, the solvent was removed under reduced pressure andthe residue obtained was acidified with 1N HCl until a pH 7 was obtainedand then aqueous citric acid solution was added until a pH 5-6 wasobtained. The aqueous layer was extracted with 10% MeOH in DCM (200mL×3), the combined organic layers were dried over anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to give therespective acid (14 g, 100%).

The above acid (14 g, 40.9 mmol) was then dissolved in DMSO (70 mL) and3-(amino methyl)-4, 6-dimethylpyridin-2(1H)-one (12.4 g, 81.9 mmol) wasadded to it. The reaction mixture was stirred at room temperature for 15minutes, then PYBOP (31.9 g, 61.4 mmol) was added and stirring wascontinued for overnight at room temperature. Upon completion of thereaction as determined by TLC, the reaction mixture was poured ontoice-cold water (700 mL), stirred for 30 minutes and the precipitatedsolid was collected by filtration, washed with water (500 mL) and airdried. The solid obtained was stirred with acetonitrile (75 mL×2),filtered and air dried. The solid obtained was again stirred with 5%MeOH in DCM (100 mL), filtered and dried completely under vacuum toafford the title compound as a solid (14 g, 74%). ¹H NMR (DMSO-d₆, 400MHz) δ 11.47 (s, 1H), 8.23 (t, 1H), 7.30 (s, 1H), 7.08 (s, 1H), 5.85 (s,1H), 4.23 (d, 2H, J=4.4 Hz), 3.81 (d, 2H, J=10.4 Hz), 3.20-3.26 (m, 2H),3.00-3.07 (m, 1H), 2.91-2.96 (m, 2H), 2.18 (s, 3H), 2.14 (s, 3H), 2.10(s, 3H), 1.58-1.60 (m, 2H), 1.45-1.50 (m, 2H), 0.78 (t, 3H, J=6.8 Hz).

Step 7: Synthesis of N-((4, 6-dimethyl-2-oxo-1, 2-dihydropyridin-3-yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1, 1′-biphenyl]-3-carboxamide

To a stirred solution of 5-bromo-N-((4, 6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl)-3-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-2-methylbenzamide (14 g, 29.5 mmol) in dioxane/water mixture (70mL/14 mL) was added 4-(4-(4, 4, 5, 5-tetramethyl-1, 3,2-dioxaborolan-2-yl) benzyl) morpholine (13.4 g, 44.2 mmol) followed byaddition of Na₂CO₃ (11.2 g, 106.1 mmol). The solution was purged withargon for 15 minutes and then Pd (PPh₃)₄ (3.40 g, 2.94 mmol) was addedand the solution was again purged with argon for a further 10 min. Thereaction mixture was heated at 100° C. for 4 h. After completion(monitored by TLC), the reaction mixture was diluted with water andextracted with 10% MeOH/DCM. The combined organic layers were dried overanhydrous sodium sulphate, filtered and concentrated under reducedpressure. The crude compound was purified by column chromatography(100-200 mesh silica gel) eluting with methanol: DCM to the titlecompound as a solid (12 g, 71%). Analytical Data: LCMS: 573.35 (M+1)+;HPLC: 99.5% (@ 254 nm) (R_(f); 3.999; Method: Column: YMC ODS-A 150mm×4.6 mm×5μ; Mobile Phase: A; 0.05% TFA in water/B; 0.05% TFA inacetonitrile; Inj. Vol: 10 μL, Col. Temp.: 30° C.; Flow rate: 1.4mL/min.; Gradient: 5% B to 95% B in 8 min, Hold for 1.5 min, 9.51-12 min5% B); ¹H NMR (DMSO-d₆, 400 MHz) δ 11.46 (s, 1H), 8.19 (t, 1H), 7.57 (d,2H, J=7.2 Hz), 7.36-7.39 (m, 3H), 7.21 (s, 1H), 5.85 (s, 1H), 4.28 (d,2H, J=2.8 Hz), 3.82 (d, 2H, J=9.6 Hz), 3.57 (bs, 4H), 3.48 (s, 2H), 3.24(t, 2H, J=10.8 Hz), 3.07-3.09 (m, 2H), 3.01 (m, 1H), 2.36 (m, 4H), 2.24(s, 3H), 2.20 (s, 3H), 2.10 (s, 3H), 1.64-1.67 (m, 2H), 1.51-1.53 (m,2H), 0.83 (t, 3H, J=6.4 Hz).

Step 8: Synthesis ofN-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamidetrihydrochloride

N-((4, 6-dimethyl-2-oxo-1, 2-dihydropyridin-3-yl) methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl) amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide (12 g, 21.0 mmol) was dissolved in methanolicHCl (200 mL) and stirred at room temperature for 3 h. After three hoursof stirring, the reaction mixture was concentrated under reducedpressure. The solid obtained was stirred with ether (100 mL×2) to affordthe desired salt as a solid (11 g, 77%). Analytical Data of the tri-HClsalt: LCMS: 573.40 (M+1)+; HPLC: 99.1% (@ 254 nm) (R_(f); 3.961; Method:Column: YMC ODS-A 150 mm×4.6 mm×5; Mobile Phase: A; 0.05% TFA inwater/B; 0.05% TFA in acetonitrile; Inj. Vol: 10 μL, Col. Temp.: 30° C.;Flow rate: 1.4 mL/min.; Gradient: 5% B to 95% B in 8 min, Hold for 1.5min, 9.51-12 min 5% B); ¹H NMR (D₂O 400 MHz) δ 7.92 (bs, 1H,) 7.80 (s,1H), 7.77 (d, 2H, J=8 Hz), 7.63 (s, 1H), 7.61 (s, 1H), 6.30 (s, 1H),4.48 (s, 2H), 4.42 (s, 2H), 4.09-4.11 (m, 4H), 3.95-3.97 (m, 2H), 3.77(t, 3H, J=10.4 Hz), 3.44-3.47 (m, 3H), 3.24-3.32 (m, 3H), 2.42 (s, 3H),2.35 (s, 3H), 2.26 (s, 3H), 2.01 (m, 2H), 1.76 (m, 2H), 1.04 (t, 3H,J=6.8 Hz).

Example 2

Combination Therapy of Compound 44 and CHOP Components

The human lymphoma cell lines WSU-DLCL2 (DSMZ; ACC 575), OCI-Ly19 (DSMZ;ACC 528), RL (ATCC; CRL-2261) were obtained from the indicated sourcesand maintained in RPMI-1640 medium supplemented with 10% fetal bovineserum and 2 mM glutamine. The cells were cultured in tissue cultureflasks in a humidified incubator at 37° C., in an atmosphere of 5% CO₂and 95% air.

The effect of combination therapy of Compound 44 with each individualCHOP (Cyclophosphamide, Vincristine, Doxorubicin, and Prednisolone)component on cancer cell viability was investigated in vitro. The dosageschedule is depicted in FIG. 1A. WSU-DLCL2 human lymphoma cells weretreated with increasing concentrations of Compound 44. After 4 days, acombination of increasing concentrations of Compound 44 and each CHOPcomponent were administered to the cells. After 4 days, cell viabilitywas determined using Millipore Guava ViaCount Reagent and flow cytometryanalysis. The percentage of viable cells for each sample was normalizedto the percentage of viable cells for the DMSO-treated samples withineach Compound 44 concentration group.

Cells treated with Compound 44 and CHOP components alone showed adecrease in cell viability. Cells treated with Compound 44 withMafofsamide (Cyclophosphamide metabolite) (FIG. 2A) and Doxorubicin(FIG. 2B) did not exhibit reduced cell viability at increasingconcentrations of Mafofsamide or Doxorubicin. Combination therapy ofCompound 44 with Vincristine (FIG. 2C) showed reduced cell viability atthe highest concentration Compound 44. Importantly, combination therapyutilizing Compound 44 and Prednisolone (Prednisone metabolite) (FIG. 2D)showed synergistic reduction in cell viability at the 2 highest doses ofCompound 44 and all doses of Prednisolone.

Example 3

Synergistic Effects of Compound 44 and Prednisolone Combination Therapyis Dependent on Dosage Schedule

To investigate the role of the timing of administration of Compound 44and Prednisolone on cell viability, WSU-DLCL2 cells were treated withdifferent dosing schedules, as depicted in FIG. 1. Cell viability wasdetermined by staining with Millipore Guava ViaCount Reagent and thenanalyzed by flow cytometry.

Administration of Compound 44 prior to co-administration of Compound 44and Prednisolone resulted in reduced cell viability (FIG. 3A). Cellstreated as depicted in FIG. 1A. Cell viability was normalized to theDMSO/DMSO sample, thereby revealing the effect of treatment withCompound 44 alone. Increasing concentrations of Compound 44 resulted ina reduction of cell growth. Importantly, increasing concentrations ofPrednisolone in combination with Compound 44 resulted in additionalreduction of cell growth compared to treating the cells with Compound 44or Prednisolone independently as single agents. Therefore, combinationtherapy of Compound 44 and Prednisolone, wherein Compound 44 isadministered first causes a synergistic effect of reducing cancer cellviability.

Administration of Compound 44 prior to administration of Prednisoloneresulted in reduced cell viability (FIG. 3B). Cells were treated asdepicted in FIG. 1B. Cell viability was normalized to the DMSO-treatedsample for each Compound 44 concentration group. Treatment withincreasing concentrations of Prednisolone as a single agent after aprior administration of Compound 44 also resulted in additionalreduction of cell viability when compared to cells treated withPrednisolone alone, thereby demonstrating the synergistic effect ofcombination therapy with Compound 44 and Prednisolone.

Administration of Prednisone prior to co-administration of Compound 44and Prednisolone did not reduce cell viability (FIG. 3C). Cells weretreated as depicted in FIG. 1C. Cell viability was normalized to theDMSO-treated sample for each Compound 44 concentration group. Theseresults demonstrated that administration of Prednisolone prior totreatment with a composition comprising Compound 44 and Prednisolone didnot cause a synergistic effect on cell viability.

These data clearly show that the combination therapy of Compound 44 andPrednisolone decreases cancer cell viability or induces cancer celldeath. Specifically, combination therapy wherein the cells are treatedwith Compound 44 prior to treatment with Prednisolone or a combinationof Prenisolone and Compound 44 results in a synergistic effect on cellviability, wherein the reduction of cell viability is greater than thatinduced by treatment of either Prednisolone or Compound 44 as singleagents.

Example 4

Compound 44 and Prednisolone Syngery is Dependent on EZH2 Mutation

Different human lymphoma cancer cell lines harboring different EZH2mutations were analyzed for their responsiveness to Compound 44 andPrednisolone. Cells were treated with dosing schedules as in FIG. 1A,where cells are first treated with Compound 44, and then after 4 days,are treated with a combination of Compound 44 and Prednisolone. Cellviability was determined 4 days later using Millipore Guava ViaCountreagent and flow cytometry analysis. Percentage of cell viability wasnormalized to percentage of the DMSO-treated sample.

Lymphoma cells expressing wild-type (WT) EZH2, OCI-LY19 cell line, areresistant to treatment with EZH2 inhibitors. Accordingly, treatment withincreasing concentrations of Compound 44 does not affect cell viability.Increasing concentrations of Prednisolone does not have an additional orsynergistic effect on cell viability (FIG. 4A).

WSU-DLCL2 cells harboring an Y641F mutation are sensitive to treatmentwith EZH2 inhibitors, as evidenced by a decrease in cell viability atincreasing concentrations of Compound 44 when administered as a singleagent. Moreover, when treated in combination with Prednisolone, thecells exhibit reduced cell viability (FIG. 4B).

RL cells harbor an Y641N mutation and are resistant to EZH2 inhibitortreatment. Administration of increasing concentrations of Compound 44alone does not result in a reduction in cell viability. However,co-administration of Compound 44 with Prednisolone resulted in asynergistic effect, wherein the cell viability was decreased greaterthan that observed when Compound 44 and Prednisolone are administered assingle agents (FIG. 4C).

Taken together, these results suggest that combination therapy withCompound 44 and Prednisolone may result in a synergistic effect inreducing cancer cell viability and increasing cancer cell death in cellsthat express a mutant EZH2. Moreover, cells that are resistant to eitherdrug, Compound 44 or Prednisolone, when administered as a single agent,become sensitive to the combination treatment and cell viability isreduced.

Example 5

Pharmacokinetic Analysis of Compound 44 and CHOP ComponentsCo-Administration In Vivo

Pharmacokinetic analysis of Compound 44 in combination with each of theCHOP components (Cyclophosphamide, Vincristine, Doxorubicin, andPrednisolone) was performed to determine the absorption or distributionof Compound 44 in vivo. Male BALB/c between 8-12 weeks old and weighing20-40 g were obtained from In vivo, Bengaluru, India. Animals wereadministered one of the CHOP components as a single agent, or incombination with Compound 44. Cyclophosphamide was administered byintraperitoneal injection at 30 mg/kg. Vincristine was administered byintravenous injection at 0.375 mg/kg. Doxorubicin was administered byintravenous injection at 2.475 mg/kg. Prednisolone was administered byoral administration at 0.15 mg/kg. Compound 44 was administered at 225mg/kg by oral administration. Plasma samples were taken at varioustimepoints over a course of 24 hours after administration.

The extraction procedure for plasma study samples or the spiked plasmacalibration standards were identical: A 25 μL sample of either studysample or spiked calibration standard was added to individualpre-labeled micro-centrifuge tubes. A volume of 100 μL of IS (Glipizide,500 ng/mL) prepared in ACN was then added to the micro-centrifuge tubesexcept in blank sample where acetonitrile was added and vortexed for 5minutes. Samples were centrifuged for 10 minutes at the speed of 15000rpm (20600 g) at 4° C. Following centrifugation, 100 μL of thesupernatant was sampled from each centrifuge tube and transferred intoinsert vials. These vials were loaded in auto-sampler for the LC/MS/MSanalysis. Calibration standards were prepared by spiking 10 μL ofanalyte (Compound 44, Cyclophosphamide, Doxorubicin, Vincristine andPrednisolone) in 190 μL of blank mouse plasma.

NonCompartmental-Analysis module in WinNonlin® (Version 5.2) was used toassess the pharmacokinetic parameters. The areas under the concentrationtime curve (AUC) were calculated by linear trapezoidal rule. The ratio(AUC_(combo)/AUC_(single)) of AUC for combination therapy of Compound 44with each component of CHOP (AUC_(combo)) to AUC of each CHOP componentalone (AUC_(single)) and indicated similar bioavailability when CHOPcomponents were administered alone or with Compound 44.

Example 6

Analysis of Compound 44 and CHOP Combination Therapy in Mouse XenograftModels

Mice

Female Fox Chase SCID® Mice (CB17/Icr-Prkdcscid/IcrIcoCrl, Charles RiverLaboratories) or athymic nude mice (Crl:NU(Ncr)-Foxnlnu, Charles RiverLaboratories) were 8 weeks old and had a body-weight (BW) range of16.0-21.1 g on day 1 of the study. The animals were fed ad libitum water(reverse osmosis 1 ppm Cl) and NIH 31 Modified and Irradiated Lab Diet®consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber.The mice were housed on irradiated Enrich-o'Cobs™ bedding in staticmicroisolators on a 12-hour light cycle at 20-22° C. (68-72° F.) and40-60% humidity. All procedures comply with the recommendations of theGuide for Care and Use of Laboratory Animals with respect to restraint,husbandry, surgical procedures, feed and fluid regulation, andveterinary care.

Tumor Cell Culture

Human lymphoma cell lines line were obtained from different sources(ATCC, DSMZ) and maintained at Piedmont as suspension cultures inRPMI-1640 medium containing 100 units/mL penicillin G sodium salt, 100g/mL streptomycin, and 25 g/mL gentamicin. The medium was supplementedwith 10% fetal bovine serum and 2 mM glutamine. The cells were culturedin tissue culture flasks in a humidified incubator at 37° C., in anatmosphere of 5% CO₂ and 95% air.

In Vivo Tumor Implantation

Human lymphoma cell lines were harvested during mid-log phase growth,and resuspended in PBS with 50% Matrigel™ (BD Biosciences). Each mousereceived 1×10⁷ cells (0.2 mL cell suspension) subcutaneously in theright flank. Tumors were calipered in two dimensions to monitor growthas the mean volume approached the desired 80-120 mm³ range. Tumor size,in mm³, was calculated from:

${{Tumor}\mspace{14mu}{volume}} = \frac{w^{2} \times l}{2}$where w=width and l=length, in mm, of the tumor. Tumor weight can beestimated with the assumption that 1 mg is equivalent to 1 mm³ of tumorvolume. After 10-30 days (depending on the cell line used) mice with108-126 mm³ tumors were sorted into treatment groups with mean tumorvolumes of 117-119 mm³.Test Articles

Compounds of Formula (IIa) were stored at room temperature and protectedfrom light. On each treatment day, a fresh compound formulations wereprepared by suspending the powders in 0.5% sodium carboxymethylcellulose(NaCMC) and 0.1% Tween® 80 in deionized water. The Compound 44 vehicle,0.5% NaCMC and 0.1% Tween® 80 in deionized water, was used to treat thecontrol groups at the same schedules. Formulations were stored away fromlight at 4° C. prior to administration.

Several chemotherapeutica were used in parallel to Epizyme compounds.Cyclophosphamide (Baxter, Lot #016591), was reconstituted to 20 mg/mLwith sterile saline and stored at 4° C. A fresh dosing solution wasprepared for each dose by dilution with saline. Doxorubicin (DoxorubicinMeiji®, Meiji Pharmaceutical Co. Ltd., 1 mg/mL) was stored at 4° C. anddiluted with saline on each treatment day. Vincristine (Hospira, Inc., 1mg/mL) was diluted with saline on each treatment day. Prednisone(Boehringer Ingelheim GmbH, 1 mg/mL) was diluted with PBS at thebeginning of each 5-day dosing cycle.

Treatment Plan

Mice were treated at compound doses ranging from 75-600 mg/kg and at TID(3 times a day every 8 h), BID (twice a day every 12 h) or QD (once aday) schedules for various amount of days days by oral gavage orinjections via the intravenous, intraperitoneal or subcutaneous routes.Each dose was delivered in a volume of 0.2 mL/20 g mouse (10 mL/kg), andadjusted for the last recorded weight of individual animals. The maximaltreatment length was 28 days.

Median Tumor Volume (MTV) and Tumor Growth Inhibition (TGI) Analysis

Treatment efficacy was determined on the last treatment day. MTV(n), themedian tumor volume for the number of animals, n, evaluable on the lastday, was determined for each group. Percent tumor growth inhibition (%TGI) can be defined several ways. First, the difference between theMTV(n) of the designated control group and the MTV(n) of thedrug-treated group is expressed as a percentage of the MTV(n) of thecontrol group:

${\%\mspace{14mu}{TGI}} = {\left( \frac{{{MTV}(n)}_{control} - {{MTV}(n)}_{treated}}{{{MTV}(n)}_{control}} \right) \times 100}$

Another way of calculating % TGI is taking the change of the tumor sizefrom day 1 to day n into account with n being the last treatment day.

${\%\mspace{14mu}{TGI}} = {\left( \frac{{\Delta\;{MTV}_{control}} - {\Delta\;{MTV}_{treated}}}{\Delta\;{MTV}_{control}} \right) \times 100}$Δ MTV_(control) = MTV(n)_(control) − MTV(1)_(control)Δ MTV_(treated) = MTV(n)_(treated) − MTV(1)_(treated)Tumor Growth Delay Analysis

Alternatively, mice were kept alive after the last treatment day fortumor growth delay analysis. Tumors were callipered twice-weekly andeach test animal was euthanized when its neoplasm reached the endpointvolume of 2000 mm³ or on the pre-specified last day of the study,whichever came first. The time-to-endpoint (TTE) for each mouse wascalculated from the following equation:

${{TTE}({days})} = \frac{{\log_{10}\left( {{{endpoint}\mspace{14mu}{volume}},{mm}^{3}} \right)} - b}{m}$where b is the intercept and m is the slope of the line obtained bylinear regression of a log-transformed tumor growth data set. The datasets were composed of the first observation that exceeded the studyendpoint volume and the three consecutive observations that immediatelypreceded the attainment of the endpoint volume. Animals that did notreach the volume endpoint were assigned a TTE value equal to the lastday of the study (prespecified). Any animal classified as atreatment-related (TR) death was to be assigned a TTE value equal to theday of death. Any animal classified as a nontreatment-related (NTR)death was excluded from TTE calculations and all further analyses.

Treatment outcome was determined from tumor growth delay (TGD), definedas the increase in the median TTE in a treatment group compared to thecontrol group:TGD=T−Cexpressed in days, or as a percentage of the median TTE of the controlgroup:

${\%\mspace{14mu}{TGD}} = {\frac{T - C}{C} \times 100}$

where:

T=median TTE for a treatment group

C=median TTE for the control group

Toxicity

Animals were weighed daily on Days 1-5, and then twice weekly until thecompletion of the study. The mice were examined frequently for overtsigns of any adverse, treatment related side effects, which weredocumented. Acceptable toxicity for the maximum tolerated dose (MTD) wasdefined as a group mean BW loss of less than 20% during the test, andnot more than 10% mortality due to TR deaths. A death was to beclassified as TR if it was attributable to treatment side effects asevidenced by clinical signs and/or necropsy, or due to unknown causesduring the dosing period. A death was to be classified as NTR if therewas evidence that the death was unrelated to treatment side effects. NTRdeaths during the dosing interval would typically be categorized as NTRa(due to an accident or human error) or NTRm (due to necropsy-confirmedtumor dissemination by invasion and/or metastasis). Orally treatedanimals that die from unknown causes during the dosing period may beclassified as NTRu when group performance does not support a TRclassification and necropsy, to rule out a dosing error, is notfeasible.

Sampling

On several days during the study mice were sampled in a pre-specifiedfashion. Sampling included non-terminal bleeds (0.25 mL) from themandibular vein without anesthesia and full volume blood collection viaterminal cardiac puncture under CO₂ anesthesia. Blood samples wereprocessed for plasma, with K₂-EDTA as anti-coagulant. The plasma sampleswere frozen at −80° C. and stored prior to bioanalysis of compoundlevels.

Tumors were harvested from specified mice under RNAse free conditionsand bisected. A 2 mm thick slice from one half of each tumor wasformalin-fixed for 24 h and transferred to 70% ethanol. The fixed tumortissues were paraffin embedded. The remaining tumor tissue from eachanimal was snap frozen in liquid N2 and pulverized with a mortar andpestle.

Specified mice were sampled for the surrogate tissues including spleen,skin, bone marrow, and whiskers. Each tissue was isolated and fixedand/or snap frozen.

Statistical and Graphical Analyses

All statistical and graphical analyses were performed with Prism 3.03(GraphPad) for Windows. Several analyses methods were applied. MedianD29 tumor volumes were compared with the Kruskal-Wallis test, and a posthoc Dunn's multiple comparison test. These tests were performed threetimes.

The two-tailed statistical analyses were conducted at P=0.05. Prismreports results as non-significant (ns) at P>0.05, significant(symbolized by “*”) at 0.01<P<0.05, very significant (“**”) at0.001<P<0.01 and extremely significant (“***”) at P>0.001.

To test statistical significance between the control and treated groupsover the whole treatment time course either a Repeated measures ANOVAtest followed by Dunnets multiple comparison post test or a 2 way ANOVAtest were employed.

For graphical representations s “box and whiskers” diagram wasconstructed to show the distribution of individual tumor volumes foreach group. The box represents the 25th 15 to 75th percentile ofobservations, the horizontal line corresponds to the median value, andthe “whiskers” indicate the maximum and minimum values. Median or mean(±SEM) tumor volumes were graphed on a semilog or linear plot asfunctions of time. Group mean BW changes during the study were plottedas percent change, ±SEM, from D1.

A scatter plot was constructed to show TTE values, by group. The TTEplot includes NTR deaths, which are excluded from all other graphicalanalyses. When an animal exited the study because of tumor size, thefinal tumor volume recorded for the animal was included with the dataused to calculate the median volume at subsequent time points. Thepercentage of animals in each group remaining in the study versus timewas presented in a Kaplan-Meier survival plot.

Histone Extraction

For isolation of histones, 60-90 mg tumor tissue was homogenized in 1.5ml nuclear extraction buffer (10 mM Tris-HCl, 10 mM MgCl2, 25 mM KCl, 1%Triton X-100, 8.6% Sucrose, plus a Roche protease inhibitor tablet1836145) and incubated on ice for 5 minutes. Nuclei were collected bycentrifugation at 600 g for 5 minutes at 4° C. and washed once in PBS.Supernatant was removed and histones extracted for one hour, withvortexing every 15 minutes, with 0.4 N cold sulfuric acid. Extracts wereclarified by centrifugation at 10000 g for 10 minutes at 4° C. andtransferred to a fresh microcentrifuge tube containing 10× volume of icecold acetone. Histones were precipitated at −20° C. for 2hours-overnight, pelleted by centrifugation at 10000 g for 10 minutesand resuspended in water.

ELISA

Histones were extracted from tumor samples as described above. Histoneswere prepared in equivalent concentrations in coating buffer (PBS+0.05%BSA) yielding 0.5 ng/ul of sample, and 100 ul of sample or standard wasadded in duplicate to 2 96-well ELISA plates (Thermo Labsystems, Immulon4HBX #3885). The plates were sealed and incubated overnight at 4° C. Thefollowing day, plates were washed 3× with 300 ul/well PBST (PBS+0.05%Tween 20; 10×PBST, KPL #51-14-02) on a Bio Tek plate washer. Plates wereblocked with 300 ul/well of diluent (PBS+2% BSA+0.05% Tween 20),incubated at RT for 2 hours, and washed 3× with PBST. All antibodieswere diluted in diluent. 100 ul/well of anti-H3K27me3 (CST #9733, 50%glycerol stock 1:1,000) or anti-total H3 (Abcam ab1791, 50% glycerol1:10,000) was added to each plate. Plates were incubated for 90 min atRT and washed 3× with PBST. 100 ul/well of anti-R_(b)-IgG-HRP (CellSignaling Technology, 7074) was added 1:2,000 to the H3K27Me3 plate and1:6,000 to the H3 plate and incubated for 90 min at RT. Plates werewashed 4× with PBST. For detection, 100 ul/well of TMB substrate (BioFxLaboratories, # TMBS) was added and plates incubated in the dark at RTfor 5 min. Reaction was stopped with 100 ul/well 1N H₂SO₄. Absorbance at450 nm was read on SpectaMax M5 Microplate reader.

Efficacy Study in SUDHL6 Xenograft Model

The efficacy of treatment with Compound 44 and in combination with CHOPon tumor growth inhibition in vivo was determined in the SUDHL6xenograft model. Comparison of tumor growth and survival rateestablished that the administration of Compound 44 and CHOP inhibited ordelayed tumor growth and increased survival of the tumor-bearing mice.Athymic nude mice were subcutaneously injected with 1×10⁷ SUDHL6 humanlymphoma cells. Compound 44 was administered once a day (QD), twice aday (BID), or three times a day (TID) at the indicated concentrations(75 mg/kg, 150 mg/kg, or 225 mg/kg). Mice received CHOP on day 1 and 8.Tumor volume was measured twice a week until the endpoint of 60 days orwhen tumor volume reached 2000 mm³, whichever occurred first.

The combination therapy of Compound 44 and CHOP showed inhibition oftumor growth over the course of treatment (28 days) in comparison tomice treated with CHOP or Compound 44 alone (FIG. 6A). Significantly,mice receiving combination therapy of Compound 44 and CHOP exhibited adurable regression of tumor size; 7 out of 12 mice demonstrated completeresponses on day 60 in the 225 mg/kg BID and CHOP combination group(FIG. 6A). A Kaplan-Meier curve was determined to show the survival ofthe mice in the study. 57% of mice that received the combination therapyof Compound 44 and CHOP survived 60 days after injection (FIG. 6B).Single agent efficacy with Compound 44 was not observed over the 28 daytreatment, however high intra-group variability may have masked thetreatment effect of Compound 44 as a single agent.

Efficacy Study in WSU-DLCL2 Xenograft Model

The efficacy of treatment with Compound 44 and in combination with CHOPon tumor growth inhibition in vivo was determined in the WSU-DLCL2xenograft model. Comparison of tumor growth established that theadministration of Compound 44 and CHOP inhibited or delayed tumor growthof the tumor-bearing mice. SCID mice were subcutaneously injected with1×10⁷ WSU-DLCL2 human lymphoma cells. Compound 44 was administered oncea day (QD), twice a day (BID), or three times a day (TID) at theindicated concentrations (150 mg/kg, 225 mg/kg, 300 mg/kg, or 600mg/kg). Mice received CHOP on day 1 and 22 (CHOP on day 1 and 8 were nottolerated in SCID mice). Tumor volume was measured twice a week untilthe endpoint of 28 days.

Compound 44 alone and in combination with CHOP therapy resulted in tumorgrowth inhibition (FIG. 7A). Administration at 150 mg/kg three times aday (TID) and 225 mg/kg twice a day (BID) of Compound 44 as a singleagent resulted in significantly smaller tumors by volume when comparedto control vehicle treated mice (p<0.05). Moreover, the combinationtherapy of Compound 44 and CHOP showed statistically significantinhibition of tumor growth in comparison to control vehicle treated mice(p<0.001). Statistical analyses were calculated using Repeated measureANOVA followed by Dunnett post test. Tumor growth inhibition was alsocalculated, revealing that treatment of tumors with Compound 44 resultedin greater tumor growth inhibition at all dosages (Table 2).Importantly, the combination treatment of Compound 44 with CHOP resultedin the greatest tumor growth inhibition.

TABLE 2 Tumor Growth Inhibition (TGI) of WSU-DLCL2 xenograft model.Group % TGI from day 1 % TGI from day 7 150 mg/kg TID 73 86 225 mg/kgBID 71 80 300 mg/kg BID 57 67 600 mg/kg QD 58 70 CHOP combo 93 100 CHOP45 51

To examine the absorption and distribution of the administered agents,pharmacokinetic analysis was performed to determine Compound 44concentration (ng/mL) in the plasma of tumor-bearing mice (FIG. 7B).Plasma samples were obtained on day 28 at 5 minutes prior to the lastdose (“trough”) and 3 hours after the last dose (“post”). Plasma sampleswere analyzed by LC-MS/MS to determine concentration Compound 44(ng/mL). Compound 44 levels were not significantly different betweenmice that received 225 mg/kg of Compound 44 twice a day (BID) as asingle agent and 225 mg/kg of Compound 44 (BID) with CHOP at the troughtimepoint. However, 3 hours post-administration of the last dose,Compound 44 levels were significantly increased in mice that received225 mg/kg of Compound 44 twice a day (BID) with CHOP compared to singleagent Compound 44.

Pharmacokinetic analysis was also performed to determine Compound 44concentration (ng/g) in tumor tissue harvested from mice on day 28 at 3hours after the last administration of treatment (FIG. 7C).

Efficacy of target inhibition in vivo was determined by analysis ofhistone methylation status in tumors after 28 days of treatment. WesternBlot analysis (FIG. 8A) demonstrated that methylation of H3K27 wasinhibited by Compound 44 at all dosages and dosing schedules inWSU-DLCL2 tumors. Histones were extracted as described above. Proteinconcentrations for acid extracted histones were determined by BCA assay(Pierce). 400-800 ng of each lysate was fractionated on 10-20%Tris-Glycine gel (Biorad), transferred using iBlot (7 minutes on program3, using Nitrocellulose transfer stacks), and probed with the followingantibodies in Odyssey blocking buffer: rabbit anti-H3K27me3 (CST 9733;1:20000 dilution) and mouse anti-Total H3 (CST 3638; 1:20000 dilution).Following primary Ab incubation, membranes were probed with IRDye 800CWDonkey-anti-mouse IgG (LiCOR #926-32212) and Alexa Fluor 680goat-anti-rabbit IgG (Invitrogen # A-21076) secondary Ab and imagedusing the LiCOR Odyssey system. Signal from total histone H3 protein wasused as control.

Analysis of histone methylation in WSU-DLCL2 tumors by ELISA confirmedthat methylation of histones was inhibited by Compound 44 at all dosagesand dosing schedules (FIG. 8B). Tumors from mice that received vehicleor CHOP did not exhibit any inhibition of methylation at H3K27. Histoneswere extracted from tumors after 28 days of treatment and analyzed byELISA as described above.

Efficacy Study in SUDHL10 Xenograft Model

The efficacy of treatment with Compound 44 and in combination with COP(Cyclosphosphamide, Oncovin [vincristine], and Prednisone) on tumorgrowth inhibition in vivo was determined in the SUDHL 10 xenograftmodel. Comparison of tumor growth established that the administration ofCompound 44 and COP inhibited or delayed tumor growth of thetumor-bearing mice. SCID mice were subcutaneously injected with 1×10⁷SUDHL10 human lymphoma cells. Compound 44 was administered twice a day(BID), or three times a day (TID) at the indicated concentrations (125mg/kg, 250 mg/kg, or 500 mg/kg). Mice received COP on day 1 and 22 (CHOPon day 1 and 8 were not tolerated in SCID mice). Tumor volume wasmeasured twice a week until the endpoint of 28 days. Half the cohortwere euthanized after 28 days of treatment, while the other half wereanalyzed until a 60 day endpoint for analysis of tumor growth delay.

FIG. 9A shows that administration of Compound 44 alone showedstatistically significant tumor growth inhibition as a single agent at250 mg/kg and 500 mg/kg when delivered twice a day (BID), when comparedto control vehicle treated mice (p<0.001). Moreover, the combinationtherapy of Compound 44 at 250 mg/kg twice a day (BID) and COP showedstatistically significant inhibition of tumor growth in comparison tocontrol vehicle treated mice (p<0.001). Statistical analyses werecalculated using Repeated measure ANOVA followed by Dunnett post test.One mouse in the 500 mg/kg BID group was euthanized on day 15 due topoor body condition. Mice receiving Compound 44 and COP combinationtherapy exhibited 8% body weight loss on day 25, in which dosing wasstopped but resumed at day 27. On day 29, 1/16 mice and 2/15 mice werewithout tumor in 250 mg/kg and 500 mg/kg group. Strikingly, 10/14 micereceiving Compound 44 and COP combination therapy were without tumor onday 29.

Tumor growth inhibition was also calculated, revealing that treatment ofSUDHL10 xenograft tumors with Compound 44, with or without COP resultedin effective tumor growth inhibition at all dosages (Table 3).

TABLE 3 Tumor Growth Inhibition (TGI) in SUDHL10 xenograft model. Group% TGI from day 1 % TGI from day 7 125 mg/kg BID 54 57 250 mg/kg BID 101113 500 mg/kg BID 104 115 COP 42 43 COP combo 107 108

Tumors were weighed after mice were euthanized on day 28 (FIG. 9B).Tumors from mice receiving single agent administration of Compound 44 at125 mg/kg or COP were significantly smaller compared to control mice(vehicle). Tumors from mice receiving combination therapy of Compound 44and COP were significantly smaller than tumors from mice receivingCompound 44 as a single agent at both 250 mg/kg and 500 mg/kg dosages.

Half the cohort was maintained to day 60, to determine efficacytreatment by determining tumor growth delay (FIG. 9C). Tumor growth inmice treated with 250 mg/kg and 500 mg/kg doses of Compound 44 as asingle agent exhibited small tumors at day 28, while control and COPtreated mice exhibited large tumors where the tumors continued to growafter day 28. Strikingly, mice receiving the combination therapy notonly had smaller tumors than all other treatment groups, but alsodisplayed significant and durable tumor growth delay.

FIG. 9D shows a Kaplan-Meier curve depicting the survival rate of micetreated with Compound 44 alone or in combination in COP. Administrationof Compound 44 as a single agent at 125 mg/kg, 250 mg/kg, and 500 mg/kgincreased the survival of mice compared to control mice or mice treatedwith COP only. Significantly, combined administration of Compound 44with COP improved the survival rate of mice compared to single agentadministration.

Pharmacokinetic and pharmacodynamic studies were performed on theSUDHL10 xenograft model. FIG. 10A shows pharmacokinetic analysis ofCompound 44 concentrations in plasma levels (ng/mL). Plasma samples wereobtained from tumor-bearing mice on day 28 at either 5 minutes prior tothe last dose (“trough”) or 3 hours after the last dose (“post”). Levelsof Compound 44 was determined by LC-MS. Pharmacodynamic analysis wasperformed by ELISA and determining the ratio of tri-methylated H3K27 intumor samples. Pharmacodynamic analysis was also performed in othertissues, specifically the spleen and bone marrow, of the tumor-bearingmice to show the efficacy of inhibition of histone methylation byCompound 44 in normal tissues of the surrogate mouse (FIGS. 10B and10C).

Example 7

Synergistic Effects of Compound 44 and Anti-Cancer Agents CombinationTherapy

Methods

The human lymphoma cell lines WSU-DLCL2 (DSMZ; ACC 575), SU-DHL-10(DSMZ; ACC 576), and Toledo (ATCC; CRL-2631) were obtained from theindicated sources and maintained in RPMI-1640 media supplemented with10%-20% heat inactivated fetal bovine serum and 2 mM glutamine. Thecells were cultured in tissue culture flasks in a humidified incubatorat 37° C., in an atmosphere of 5% CO₂ and 95% air. The WSU-DLCL2 andSU-DHL-10 contain the Y641 EZH2 mutation, and the Toledo cell linecontains WT EZH2.

The in vitro anti-proliferative effects of the combination of Compound44 with the following drugs: AraC, Cisplatin, Decitabine, Dexamethasone,Everolimus, Prednisolone, and Doxorubicin were investigated. WSU-DLCL2,SU-DHL-10, or Toledo human lymphoma cells were treated with increasingconcentrations of Compound 44 in flasks. After 4 days of treatment, theWSU-DLCL2 or SU-DHL-10 cells were split to initial seeding density andplated into a 96-well tissue culture plate with each concentration ofCompound 44 in one row of the plate. After 6 days the Toledo cells weresplit to initial seeding density and plated into a 96-well tissueculture plate with each concentration of Compound 44 in one row of theplate. Increasing doses of drugs were then added to the plates. One doseper column forming a matrix of Compound 44 and drug doses. Afterincubation for an additional 3 days cell viability of WSU-DLCL2 orSU-DHL-10 was measured using Promega Cell Titer-Glo reagent followed byluminescence detection. After 5 days viability of Toledo cells wasmeasured using Promega Cell Titer-Glo reagent followed by luminescencedetection.

Data Analysis

Synergy was determined using the software package Calcusyn by Biosoftbased on the Chou-Talalay method for drug combination which uses themedian-effect equation (Chou 2006). First, raw luminescence values wereconverted to percent inhibition or fraction affected (Fa) calculatedusing controls for maximum inhibition and DMSO treated cells for minimuminhibition control located on each plate. Percent inhibition values foreach constant ratio of compound combinations were entered into Calcusynto determine the combination index values. Combination index values lessthan 1 indicated synergy.

For test compounds that did not inhibit cells viability by 50%, synergycannot be determined using Calcusyn. Instead, the data was reported asfold IC50 shift of the IC50 for Compound 44. Alternatively, if Compound44 did not have an effect, as with the Toledo cells, the fold IC50 shiftwas reported for the drug instead of Compound 44.

Results

Treatment of WSU-DLCL2 and SU-DHL-10 cells with Compound 44 and eitherAraC, Cisplatin, Doxorubicin, Decitabine, or Everolimus demonstratedsynergistic reduction in cell viability. Combination index values forcombinations with Compound 44 and Decitabine in WSU-DLCL2 and SU-DHL-10cells were below 0.1 which is denoted by very strong synergism accordingto the Chou-Talalay characterization (Chou 2006). Combination indexvalues for combinations with Compound 44 and Everolimus in WSU-DLCL2cells were below 0.1 and SU-DHL-10 cells between 0.1-0.3 which isdenoted by very strong synergism and strong synergism respectively.Combinations with Compound 44 and either AraC, Cisplatin, or Doxorubicinin WSU-DLCL2 and SU-DHL-10 cells had combination index values between0.3-0.7 which denotes synergism (Chou 2006). Combination of Compound 44with Prednisolone enhanced the Compound 44 potency by 7-fold forWSU-DLCL2 and 3-fold for SU-DHL-10 cells at the top dose ofPrednisolone. Additionally, combination with Compound 44 andDexamethasone enhanced the Compound 44 potency by 17-fold for WSU-DLCL2cells and 3-fold for SU-DHL-10 cells at the top does of Dexamethasone.

The Toledo cell line showed no sensitivity to Compound 44 and nocombination benefit was seen when Compound 44 was combined with eitherAraC, Cisplatin, Doxorubicin, Decitabine, Everolimus, Prednisolone, orDexamethasone. Thus the combination benefit seen in these experimentsappears to be dependent on EZH2 mutation.

TABLE 4 List of compounds and cell lines WSU SUDHL10 Toledo Prednisolone7-fold 3-fold No enhancement enhancement enhancement DoxorubicinSynergism Synergism No enhancement CI 0.3-0.7 CI 0.3-0.7 CisplatinSynergism n/a No enhancement CI 0.3-0.7 AraC Synergism Synergism Noenhancement CI 0.3-0.7 CI 0.3-0.7 Everolimus Very strong Strong Noenhancement synergism synergism CI < 0.1 CI 0.1-0.3 Decitabine Verystrong Very strong No enhancement synergism synergism CI < 0.1 CI < 0.1Dexamethasone 17-fold 3-fold No enhancement enhancement enhancement

INCORPORATION BY REFERENCE

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EQUIVALENTS

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A method of treating follicular lymphomacomprising administering to a subject in need thereof a therapeuticallyeffective dose of a compound of Formula (IIa):

or a pharmaceutically acceptable salt thereof and a therapeuticallyeffective dose of rituximab, wherein each of R_(a) and R_(b),independently, is H or C₁-C₆ alkyl; or R_(a) and R_(b), together withthe N atom to which they are attached, form a 4 to 7-memberedheterocycloalkyl ring having 0 or 1 additional heteroatoms; wherein theC₁-C₆ alkyl or the 4 to 7-membered heterocycloalkyl ring are optionallysubstituted with one or more -Q₃-T₃, in which Q₃ is a bond orunsubstituted or substituted C₁-C₃ alkyl linker, and T₃ is H, halo, 4 to7-membered heterocycloalkyl, C₁-C₃ alkyl, OR_(d), COOR_(d), —S(O)₂R_(d),or —NR_(d)R_(e), in which each of R_(d) and R_(e) is independently H orC₁-C₆ alkyl, or -Q₃T₃ is oxo; R₇ is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, or 4to 12-membered heterocycloalkyl, each optionally substituted with one ormore -Q₅-T₅, in which Q₅ is a bond, C(O), C(O)NR_(k), NR_(k)C(O), S(O)₂,or C₁-C₃ alkyl linker, R_(k) being H or C₁-C₆ alkyl, and T₅ is H, halo,C₁-C₆ alkyl, hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to12-membered heterocycloalkyl, ₅- or 6-membered heteroaryl, orS(O)_(q)R_(q) in which q is 0, 1, or 2 and R_(q) is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl, and T₅ is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, C₁-C₆ alkyl, hydroxyl, cyano, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-membered heteroarylexcept when T₅ is H, halo, hydroxyl, or cyano; or -Q₅-T₅ is oxo; and R₈is H, methyl, or ethyl; wherein the follicular lymphoma is resistant orrefractory to at least one prior therapy; and wherein the compound ofFormula (IIa) and said rituximab are administered simultaneously orsequentially.
 2. The method of claim 1, wherein the compound of Formula(IIa) or a pharmaceutically acceptable salt thereof, is administeredprior to administration of said rituximab.
 3. The method of claim 1,wherein said subject has demonstrated resistance to the compound ofFormula (IIa) or a pharmaceutically acceptable salt thereof whenadministered as a single agent.
 4. The method of claim 1, wherein thefollicular lymphoma is resistant to at least one prior monotherapy. 5.The method of claim 1, wherein the follicular lymphoma is resistant toat least one prior combination therapy.
 6. The method of claim 1,wherein said subject has demonstrated resistance to rituximab whenadministered as a single agent.
 7. The method of claim 1, wherein thecompound of Formula (IIa) is Compound 44:

or a pharmaceutically acceptable salt thereof.
 8. The method of claim 1,wherein R₇ is a C₁-C₆ alkyl, a 4 to 12-membered heterocycloalkyl, or aC₃-C₈ cycloalkyl, wherein the C₁-C₆ alkyl is isopropyl, the 4 to12-membered heterocycloalkyl is selected from piperidinyl,tetrahydropyran, and tetrahydro-2H-thiopyranyl, and the C₃-C₈ cycloalkylis selected from cyclopentyl and cyclohexyl.
 9. A method of treatingcancer comprising administering to a subject in need thereof atherapeutically effective dose of a compound of Formula (IIa):

or a pharmaceutically acceptable salt thereof, a therapeuticallyeffective dose of rituximab, and a therapeutically effective dose oflenalidomide, wherein each of R_(a) and R_(b), independently, is H orC₁-C₆ alkyl; or R_(a) and R_(b), together with the N atom to which theyare attached, form a 4 to 7-membered heterocycloalkyl ring having 0 or 1additional heteroatoms; wherein the C₁-C₆ alkyl or the 4 to 7-memberedheterocycloalkyl ring are optionally substituted with one or more-Q₃-T₃, in which Q₃ is a bond or unsubstituted or substituted C₁-C₃alkyl linker, and T₃ is H, halo, 4 to 7-membered heterocycloalkyl, C₁-C₃alkyl, OR_(d), COOR_(d), —S(O)₂R_(d), or -NR_(d)R_(e), in which each ofR_(d) and R_(e) is independently H or C₁-C₆ alkyl, or -Q₃T₃ is oxo; R₇is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, or 4 to 12-membered heterocycloalkyl,each optionally substituted with one or more -Q₅-T₅, in which Q₅ is abond, C(O), C(O)NR_(k), NR_(k)C(O), S(O)₂, or C₁-C₃ alkyl linker, R_(k)being H or C₁-C₆ alkyl, and T₅ is H, halo, C₁-C₆ alkyl, hydroxyl, cyano,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, ₅- or6-membered heteroaryl, or S(O)_(q)R_(q) in which q is 0, 1, or 2 andR_(q) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or 6-memberedheteroaryl, and T₅ is optionally substituted with one or moresubstituents selected from the group consisting of halo, C₁-C₆ alkyl,hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl except when T₅ is H,halo, hydroxyl, or cyano; or -Q₅-T₅ is oxo; and R₈ is H, methyl, orethyl; wherein the cancer is resistant or refractory to at least oneprior therapy; and wherein the compound of Formula (IIa) and saidrituximab and said lenalidomide are administered simultaneously orsequentially.
 10. The method of claim 9, wherein the compound of Formula(IIa) or a pharmaceutically acceptable salt thereof, is administeredprior to administration of said rituximab, said lenalidomide, or acombination of said rituximab and said lenalidomide.
 11. The method ofclaim 9, wherein the cancer is resistant to at least one priormonotherapy.
 12. The method of claim 9, wherein said subject hasdemonstrated resistance to the compound of Formula (IIa) or apharmaceutically acceptable salt thereof when administered as a singleagent.
 13. The method of claim 9, wherein the cancer is resistant to atleast one prior combination therapy.
 14. The method of claim 9, whereinsaid subject has demonstrated resistance to lenalidomide whenadministered as a single agent.
 15. The method of claim 9, wherein saidsubject has demonstrated resistance to rituximab when administered as asingle agent.
 16. The method of claim 9, wherein said subject hasdemonstrated resistance to rituximab and to lenalidomide whenadministered as single agents or in combination.
 17. The method of claim9, wherein the cancer is lymphoma, leukemia, or melanoma.
 18. The methodof claim 17, wherein the cancer is follicular lymphoma.
 19. The methodof claim 9, wherein the compound of Formula (IIa) is Compound 44:

or a pharmaceutically acceptable salt thereof.
 20. The method of claim9, wherein R₇ is a C₁-C₆ alkyl, a 4 to 12-membered heterocycloalkyl, ora C₃-C₈ cycloalkyl, wherein the C₁-C₆ alkyl is isopropyl, the 4 to12-membered heterocycloalkyl is selected from piperidinyl,tetrahydropyran, and tetrahydro-2H-thiopyranyl, and the C₃-C₈ cycloalkylis selected from cyclopentyl and cyclohexyl.